Flagellin related polypeptides and uses thereof

ABSTRACT

The use of flagellin and flagellin related polypectides for the protection of mammals from the effects of apoptsis is described.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a divisional of U.S. patent application Ser. No. 11/722,682,filed on May 2, 2008, which is the national stage of InternationalApplication No. PCT/US2005/046485, filed on Dec. 22, 2005, which claimsthe benefit of U.S. Provisional Patent Application No. 60/639,826, filedDec. 22, 2004, the contents of all of which is incorporated herein byreference.

FIELD OF THE INVENTION

This invention relates to the use of flagellin related polypeptides toprotect mammals from the effects of apoptosis. More specifically, thisinvention relates to the use of flagellin related polypeptides toprotect mammals from exposure to stress, such as radiation and cancertreatments.

REFERENCE TO THE SEQUENCE LISTING

Reference is made to the sequence listing submitted via EFS-Web, whichconsists of a file named, “Sequence.txt” (134 KB), created on Jul. 1,2010, the contents of which are incorporated herein by reference.

BACKGROUND OF THE INVENTION

The progression from normal cells to tumor cells involves a loss ofnegative mechanisms of growth regulation, including resistance to growthinhibitory stimuli and a lack of dependence on growth factors andhormones. Traditional cancer treatments that are based on radiation orcytotoxic drugs rely on the differences in growth control of normal andmalignant cells. Traditional cancer treatments subject cells to severegenotoxic stress. Under these conditions, the majority of normal cellsbecome arrested and therefore saved, while tumor cells continue todivide and die.

However, the nature of conventional cancer treatment strategy is suchthat normal rapidly dividing or apoptosis-prone tissues are at risk.Damage to these normal rapidly dividing cells causes the well-known sideeffects of cancer treatment (sensitive tissues: hematopoiesis, smallintestine, hair follicles). The natural sensitivity of such tissues iscomplicated by the fact that cancer cells frequently acquire defects insuicidal (apoptotic) machinery and those therapeutic procedures thatcause death in normal sensitive tissues may not be that damaging tocancer cells. Conventional attempts to minimize the side effects ofcancer therapies are based on (a) making tumor cells more susceptible totreatment, (b) making cancer therapies more specific for tumor cells, or(c) promoting regeneration of normal tissue after treatment (e.g.,erythropoietin, GM-CSF, and KGF).

There continues to be a need for therapeutic agents to mitigate the sideeffects associated with chemotherapy and radiation therapy in thetreatment of cancer. This invention fulfills these needs and providesother related advantages.

SUMMARY OF THE INVENTION

A method of protecting a mammal from one or more treatments orconditions that trigger apoptosis comprising administering to saidpatient a composition comprising a pharmaceutically effective amount offlagellin. The flagellin may comprise SEQ ID NO: 1 or a fragment,variant, analog, homolog, derivative of SEQ ID NO: 1, or combinationthereof. The flagellin may induce TLR-5 mediated activity.

The flagellin may be at least 30% identical to amino acids 1-174 and418-505 of SEQ ID NO: 1. The flagellin may comprise at least 10conserved amino acids at positions selected from the group consisting of89, 90, 91, 95, 98, 101, 115, 422, 423, 426, 431, 436 and 452. Theflagellin may comprise the sequence of SEQ ID NOS: 1, 8, 10, 12, 30, 32,34, 36, 38, 40, 43, 44, 46, 48, 50 and 52.

The flagellin may be used to treat a mammal undergoing cancer treatment,which may be chemotherapy or radiation therapy. The flagellin may beused to treat a mammal exposed to radiation. The flagellin may beadministered in combination with a radioprotectant. The flagellin may beused to treat a mammal from wounding, poisoning, bacterial infection,viral infection and temperature shock. The flagellin may be used toprotect from apoptosis in tissues including the GI tract, lungs,kidneys, liver, cardiovascular system, blood vessel endothelium, centraland peripheral neural system, hematopoietic progenitor cells, immunesystem, and hair follicles. The flagellin may also be used to preventsepsis in the mammal.

This invention also relates to a method of treating a mammal sufferingfrom a constitutively active NF-κB cancer comprising administering tothe mammal a composition comprising a pharmaceutically acceptable amountof an agent which induces NF-κB. The agent may be flagellin. The agentmay be administered prior to, together with, or after a treatment forthe cancer. The treatment may be chemotherapy or radiation therapy.

This invention also relates to a method of treating a mammal sufferingfrom damage to normal tissue attributable to treatment of a cancercomprising administering to the mammal a composition comprising apharmaceutically acceptable amount of an agent which induces NF-κB. Theagent may be flagellin. The agent may be administered prior to, togetherwith, or after a treatment for the cancer. The treatment may bechemotherapy or radiation therapy.

This invention also relates to a method of treating a mammal sufferingfrom damage to normal tissue attributable to stress, comprisingadministering to the mammal a composition comprising a pharmaceuticallyacceptable amount of an agent which induces NF-κB. The agent may beflagellin. The agent may be administered prior to, together with, orafter a treatment for a disease suffered by the mammal.

This invention also relates to a method of modulating cell aging in amammal, comprising administering to the mammal a composition comprisinga pharmaceutically acceptable amount of an agent which induces NF-κB.The agent may be flagellin. The agent may be administered prior to,together with, or after a treatment for a disease suffered by themammal.

This invention also relates to a pharmaceutical composition comprisingan agent which induces NF-κB activity, a chemotherapeutic drug, andoptionally a pharmaceutically acceptable adjuvant, diluent, or carrier.The agent may be flagellin.

This invention also relates to a method of screening for an inducer ofNF-κB comprising adding a suspected inducer to an NF-κB activatedexpression system, and separately adding a control to an NF-κB activatedexpression system, whereby an inducer of NF-κB is identified by theability to increase the level of NF-κB activated expression.

This invention also relates to a method of protecting a mammal from theeffects of radiation comprising administering to said mammal acomposition comprising a pharmaceutically effective amount of an agentwhich induces NF-κB. The agent may be flagellin, which may be derivedfrom a species of Salmonella. The composition may be administered incombination with a radioprotectant. The radioprotectant may be anantioxidant, which may be amifostine or vitamine E. The radioprotectantmay also be a cytokine, which may be stem cell factor.

This invention relates to a method of protecting a patient from one ormore treatments or conditions that trigger apoptosis comprisingadministering to said patient a composition comprising apharmaceutically effective amount of an agent which induces NF-κB. Theagent may be flagellin, which may be derived from a species ofSalmonella. The treatment may be a cancer treatment, which may bechemotherapy or radiation therapy. The condition may be a stress, whichmay be radiation, wounding, poisoning, infection and temperature shock.

This invention also relates to a method of screening for a modulator ofapoptosis comprising adding a suspected modulator to a cell-basedapoptosis system, and separately adding a control to a cell-basedapoptosis system, whereby a modulator of apoptosis is identified by theability to alter the rate of apoptosis, wherein the suspected modulatoris derived from a mammalian parasite or symbiont.

This invention also relates to a method of screening for a modulator ofNF-κB comprising adding a suspected modulator to an NF-κB activatedexpression system, and separately adding a control to an NF-κB activatedexpression system, whereby a modulator of NF-κB is identified by theability to alter the rate of NF-κB activated expression, wherein thesuspected modulator is derived from a mammalian parasite. The parasitemay be of a species including, but not limited to, Salmonella,Mycoplasma, and Chlamydia.

This invention also relates to a modulator identified by any of thescreening methods described herein. This invention also relates to acomposition comprising a modulator described herein. The composition maybe a pharmaceutical composition comprising a pharmaceutically acceptableamount of a modulator described herein.

This invention also relates to a method of treating cancer comprisingadministering to a subject in need of such treatment a pharmaceuticalcomposition comprising a modulator that enhances apoptosis.

This invention also relates to a method of protecting a patient from oneor more treatments that trigger apoptosis comprising administering tosaid patient a pharmaceutical composition comprising a modulator thatinhibits apoptosis. The one or more treatments may be a cancertreatment. The cancer treatment may be chemotherapy or radiationtherapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 demonstrates that p53 deficiency accelerated development of GIsyndrome in mice. Panel A: I.P. injection of PFTc (10 mg/kg) protectsC57Bl/6J mice (if not indicated otherwise, here and below 6-8 weeks oldmales were used) from a single 9 Gy dose of gamma radiation and afractioned cumulative radiation dose 12.5 Gy (5×2.5 Gy). PFTc has noeffect on survival of mice treated with single 12.5 and 25 Gy doses ofIR: (results of representative experiments are shown; Shepherd 4000 CiCesium 137 source at a dose rate of 4 Gy per minute was used). Panel B:Wild-type and p53-null C57Bl/6J mice differ in their relativesensitivity to low (10 Gy) and high (15Gy) doses of gamma radiation:wild-type mice were more sensitive to 10 Gy but more resistant to 15 Gyas compared to p53-null mice. Panel C: Mice treated with 11 Gy of totalbody gamma irradiation were injected 12 h later with 1.5×10⁷ bone marrowcells from wild type or p53-null syngeneic C57Bl/6J mice. (This dosecauses 100% lethality in nonreconstituted controls group of mice). Twomonths later, after complete recovery of hematopoiesis, animals weretreated with 15 Gy of total body gamma radiation and showed nodifference in death rates between the two groups differing in the p53status of their bone marrow. Panel D: Comparison of dynamics of injuryto small intestines of wild-type and p53-null mice at the indicated timepoints after 15 Gy of gamma radiation indicates accelerated damage inp53-null mice (haematoxylin-eosin stained paraffin sections;magnification ×125). 24 h panels include images of TUNEL staining ifsections of crypts: massive apoptosis is evident in wild type but not inp53-deficient epithelium.

FIG. 2 demonstrates the dynamics of cell proliferation and survival insmall intestine of wild type and p53-null mice. Panel A: Comparison ofproliferation rates in intestines of wild-type and p53 null mice aftertreatment with IR. (Left) Autoradiographs of whole-body sections (1.7×magnification) of 4-week-old wild-type and p53 null mice injectedintraperitoneally with ¹⁴C-thymidine (10 μCi per animal) treated oruntreated with 15 Gy of gamma radiation. Arrows point at intestines.(Right) Comparison of BrdU incorporation in small intestine of wild-typeand p53-null mice at different time points after 15 Gy of gammaradiation. BrdU (50 mg/kg) was injected 2 h before sacrificing micefollowed by immunostaining. Fragments of 96 h panels are shown at highermagnification (×400). Panel B: Comparison of the number of BrdU positivecells/crypt in small intestine of wild-type and p53-null mice atdifferent time points after 15 Gy of gamma radiation. Three animals wereanalyzed for each time point, five ileum cross sections were preparedfrom each animal and analyzed microscopically to estimate the number ofcrypts and villi. Numbers of BrdU-positive cells in the crypts werecounted in 5 random fields under 200× magnification (100-30 crypts) andthe average number of BrdU-positive cells was plotted. Panel C: Tracingthe number and position of BrdU-labeled cells in small intestine of wildtype and p53-null mice during different time points after 15 Gy of gammaradiation. BrdU was injected 30 min. before irradiation and mice weresacrificed at the indicated time points. Accelerated migration fromcrypts to villi followed by rapid elimination of labeled cells wasobserved in p53-null mice.

FIG. 3 demonstrates that recombinant flagellin is capable of NF-κBactivation.

FIG. 4 shows a representative experiment testing the ability offlagellin to protect mice from radiation. C56BL6 mice (6 week old males,10 animals per group) were injected i.v. with 2.0 μg (0.1 mg/kg) or 5 μg(0.25 mg/kg) of flagellin in PBS. Four hours later, mice were irradiatedwith 15 Gy and mouse survival was monitored daily.

FIG. 5 shows histological sections (HE stained) of small intestinalepithelium of mice that were treated with 15 Gy of gamma radiation withor without i.v. injection of 0.25 mg/kg of flagellin. Completedestruction of crypts and villi in control mouse contrasts with close tonormal morphology of tissue from flagellin-treated animal.

FIG. 6 shows the effect of flagellin on mouse sensitivity to 10Gy oftotal body gamma radiation.

FIG. 7 shows the effect of flagellin injected i.v. at indicated timesbefore irradiation on mouse sensitivity to 13Gy (left) and 10Gy (right)of total body gamma radiation.

FIG. 8 shows the effect of flagellin on mouse sensitivity to 10, 13 and15 Gy of total body gamma radiation.

FIG. 9 shows the domain structure of bacterial flagellin. The Cabackbone trace, hydrophobic core distribution and structural informationof F41. Four distinct hydrophobic cores that define domains D1, D2a, D2band D3. All the hydrophobic side-chain atoms are displayed with the Cabackbone. Side-chain atoms are color coded: Ala, yellow; Leu, Be or Val,orange; Phe and Tyr, purple (carbon atoms) and red (oxygen atoms). c,Position and region of various structural features in the amino-acidsequence of flagellin. Shown are, from top to bottom: the F41 fragmentin blue; three b-folium folds in brown; the secondary structuredistribution with α-helix in yellow, b-structure in green, and b-turn inpurple; tic mark at every 50th residue in blue; domains D0, D1, D2 andD3; the axial subunit contact region within the proto-element in cyan;the well-conserved amino-acid sequence in red and variable region inviolet; point mutations in F41 that produce the elements of differentsupercoils. Letters at the bottom indicate the morphology of mutantelements: L (D107E, R124A, R124S, G426A), L-type straight; R (A449V),R-type straight; C (D313Y, A414V, A427V, N433D), curly33.

FIG. 10 shows a schematic of Salmonella flagellin domains, itsfragments, and its interaction with TLR5. Dark bars denote regions ofthe flagellin gene used to construct fragments comprising A, B, C, A′and B′.

FIG. 11 shows soluble flagellin (FliC), and two fragments (AA′ and BB′)after fractionation by SDS-PAGE, with molecular weight markers listed tothe left.

FIG. 12 shows induction of NF-κB nuclear translocation by Salmonellaflagellin (FliC) and flagellin fragments.

FIG. 13 shows activation of NF-κB-regulated luciferase reporterconstruct by flagellin and flagellin fragments in H116 cells.Concentrations of proteins are given in μg/ml.

FIG. 14 shows NF-κB DNA binding in HT29 human colon cancer cells inducedby flagellin and flagellin fragments.

FIG. 15 shows the activation of a NF-κB reporter in HCT116 reportercells by full-length flagellin and flagellin fragments.

FIG. 16 shows a comparison of the radioprotective properties offlagellin (FliC) and fragments AA′ and BB′.

FIG. 17 shows that the AA′ fragment protects intestinal epithelium fromdegeneration caused by radiation. A: Histological sections (hematoxylinand eosin-stained) of small intestinal epithelium of mice 5 days after14 Gy irradiation are shown. B: Treatment with the AA′ fragment preventsapoptosis ongoing 5 hours after irradiation in endothelial cells ofvilli (detected by immunostaining for endothelial marker CD31 and markedby arrows), as determined by TUNEL assay. C: Histological sections ofskin of mice 5 days after 14Gy of gamma irradiation demonstrate theprotective effect of the AA′ fragment for sebaceous glands (red arrows).

FIG. 18 shows that the AA′ fragment provides partial protection anddelays death of mice after supralethal irradiation with 17 and 20 Gytotal-body gamma radiation.

FIG. 19 shows anti-flagellin antibody titers induced in mice after 21and 28 days by flagellin and AA′. For individual mice, the averages oftwo measurements are shown. Mice were injected with: F1: flagellin; orAA′. 21d and 28ds—mice injected with first dose 21 and 28 days beforesecond, respectively. PBS: saline buffer (no serum) control; blank:empty well reading control.

FIG. 20 shows anti-flagellin antibody titers induced in mice after 21and 28 days by flagellin and AA′. For individual mice, the averages oftwo measurements are shown. Mice were injected with: F1: flagellin; orAA′. 21d and 28ds—mice injected with first dose 21 and 28 days beforesecond, respectively. PBS: saline buffer (no serum) control; blank:empty well reading control.

FIG. 21 shows that flagellin fragment AA′ protects mice from multiplesuccessive doses of gamma-irradiation. Arrows denote radiationtreatments (days 1-4).

FIG. 22 shows the effect of AA′ on tumor sensitivity to radiationtreatment. Left Panel: NIH3T3-derived sarcoma cells were injected s.c.in NIH-Swiss mice. When tumors reached 7-10 mm in diameter, micereceived three 4.3 Gy doses of total body irradiation, with or withoutpretreatment with AA′. The dynamics of tumor growth after radiationtreatment is displayed. U/t: untreated; AA′: AA′ with no irradiation;3×4Gy: irradiation only; 3×4Gy+AA′: AA′ and irradiation. (The shape ofcurves reflects slow growth of tumors that is a characteristic of thismodel). Results are displayed as relative tumor volumes normalized totumor volume measured at day 7 after last irradiation. Right Panel: Theexperiment was done in the same way with another syngeneic mouse tumormodel: B16 melanoma (C57BL6 background). Treatment was applied whentumors reached 4-5 mm in diameter and involved three subsequent 4 Gydoses of total body gamma radiation applied with or without pretreatmentwith AA′ (30 min. before irradiation, 5 μg/mouse).

FIG. 23 shows the influence of NS398 on the radioprotection of LPS andAA′ in mice after 13 Gy of total-body gamma irradiation.

FIGS. 24A and 24B show a comparison of amino acid sequences of theconserved amino (FIG. 24A) and carboxy (FIG. 24B) terminus from 21species of bacteria. The 13 conserved amino acids important for TLR5activity are shown with shading. The amino acid sequences are identifiedby their accession numbers from TrEMBL (first letter=Q) or Swiss-Prot(first letter=P). The amino terminus sequences have SEQ ID NOs: 1-21,respectively, for each of the 21 bacterial species, and the carboxyterminus sequences have SEQ ID NOs: 22-42, respectively.

FIG. 25 shows results of a BLAST search using SEQ ID NO: 1 as the querysequence. The parameters used in all searches was as follows: expectedvalue cutoff=10, matrix=BLOSUM62, gap penalties of existence=11 andextension=1, filtering=none. FIG. 25A: NR_Bacteria (Protein-Protein);FIG. 25B: NR_Bacteria (Protein-DNA); FIG. 25C: Bacterial Genomes(Protein-Protein); FIG. 25D: Bacterial Genomes (Protein-DNA).

FIG. 26 shows the percentage identities of the amino- andcarboxy-terminus of the homologs shown in FIG. 24 compared to SEQ ID NO:1, as shown in BLAST results using the same search parameters as listedfor FIG. 25.

FIG. 27 demonstrates that AA′ mediates rescue of multiple mouse strainsafter 10 Gy total-body γ-IR. Cone heights represent fractions ofsurvivors.

FIG. 28 demonstrates the pharmacokinetics of AA′ after intravenous(i.v.), subcutaneous (s.c.), intraperitoneal (i.p.) or intramuscular(i.m.) injection.

FIG. 29 demonstrates the extended pharmacokinetics of AA′ afterintramuscular (i.m.) injection.

FIG. 30 demonstrates the influence of AA′ on gamma-irradiation inducedcell death and growth inhibition in A549 cells.

FIG. 31 demonstrates the influence of AA′ on gamma-irradiation inducedcell death and growth inhibition in multiple cell lines.

FIG. 32 demonstrates the influence of irradiation and AA′ on BrdUincorporation in small intestinal crypts of NIH-Swiss mice. A comparisonof BrdU incorporation in small intestine of control and AA′ treatedNIH-Swiss mice, with and without 15 Gy of gamma radiation is shown. BrdU(50 mg/kg) was injected 1.5 h before sacrificing mice and immunostainingwas done as previously described (Watson A J & Pritchard D M., Am JPhysiol Gastrointest Liver Physiol. 2000 January; 278(1):G1-5). Redchannel of the image is shown (positive signal is bright white on thedark background).

FIG. 33 demonstrates the duration of AA′-mediated growth arrest andreduced BrdU incorporation in small intestine of mice. BrdU (50 mg/kg)was injected in Balb/c mice i.p., 1 or 4 hrs after CBLB502 (AA′)injection. Samples of small intestine were obtained 1.5 hrs after BrdUinjection. Immunostaining was done as previously described (Watson A J &Pritchard D M., Am J Physiol Gastrointest Liver Physiol. 2000 January;278(1):G1-5). Inverted image is shown (positive signal is dark on thelight background).

FIG. 34 demonstrates the influence of AA′ on BrdU incorporation incolonic crypts of NIH-Swiss mice. BrdU (50 mg/kg) was injected inNIH-Swiss mice i.p., 1 hr after CBLB502 (AA′) injection, Samples ofcolon were obtained 1.5 hrs after BrdU injection. Immunostaining wasdone as previously described (Watson A J & Pritchard D M., Am J PhysiolGastrointest Liver Physiol. 2000 January; 278(1):G1-5). Inverted imageis shown (positive signal is dark on the light background). Bottom panelshows smaller magnification/larger area of the sample.

FIG. 35 demonstrates the morphology of small intestine in TLR5 deficientMOLF/Ei and TLR5 wt NIH-Swiss mice after treatment with AA′.

FIG. 36 depicts flagellin derivatives. The domain structure andapproximate boundaries (amino acid coordinates) of selected flagellinderivatives (listed on the right). FliC flagellin of Salmonella dublinis encoded within 505 amino acids (aa).

FIG. 37 shows the testing of additional flagellin derivatives tested forNF-κB stimulating activity.

FIG. 38 shows the nucleotide and amino acid sequence for the followingflagellin variants: AA′ (SEQ ID NO: 7-8), AB′ (SEQ ID NO: 9-10), BA′(SEQ ID NO: 11-12), BB′ (SEQ ID NO: 13-14), CA′ (SEQ ID NO: 15-16), CB′(SEQ ID NO: 17-18), A (SEQ ID NO: 19-20), B (SEQ ID NO: 21-22), C (SEQID NO: 23-24), GST-A′ (SEQ ID NO: 25-26), GST-B′ (SEQ ID NO: 27-28),AA′n1-170 (SEQ ID NO: 29-30), AA′n1-163 (SEQ ID NO: 33-34), AA′n54-170(SEQ ID NO: 31-32), AA′n54-163 (SEQ ID NO: 335-36), AB′n1-170 (SEQ IDNO: 37-38), AB′n1-163 (SEQ ID NO: 39-40), AA′n1-129 (SEQ ID NO: 41-42),AA′n54-129 (SEQ ID NO: 43-44), AB′n1-129 (SEQ ID NO: 45-46), AB′n54-129(SEQ ID NO: 47-48), AA′n1-100 (SEQ ID NO: 49-50), AB′n1-100 (SEQ ID NO:51-52), AA′n1-70 (SEQ ID NO: 53-54) and AB′n1-70 (SEQ ID NO: 55-56). ThepRSETb leader sequence is shown in Italic (leader includes Met, which isalso amino acid 1 of FliC). The N terminal constant domain isunderlined. The amino acid linker sequence is in Bold. The C terminalconstant domain is underlined. GST, if present, is highlighted.

DETAILED DESCRIPTION

This invention is related to protecting normal cells and tissues fromapoptosis caused by stresses including, but not limited to,chemotherapy, radiation therapy and radiation. There are two majormechanisms controlling apoptosis in the cell: the p53 pathway(pro-apoptotic) and the NF-κB pathway (anti-apoptotic). Both pathwaysare frequently deregulated in tumors: p53 is usually lost, while NF-κBbecomes constitutively active. Hence, inhibition of p53 and activationof NF-κB in normal cells may protect them from death caused by stresses,such as cancer treatment, but would not make tumor cells more resistantto treatment because they have these control mechanisms deregulated.This contradicts the conventional view on p53 and NF-κB, which areconsidered as targets for activation and repression, respectively.

This invention relates to inducing NF-κB activity to protect normalcells from apoptosis. By inducing NF-κB activity in a mammal, normalcells may be protected from apoptosis attributable to cellular stress,which occurs in cancer treatments and hyperthermia; exposure to harmfuldoses of radiation, for example, workers in nuclear power plants, thedefense industry or radiopharmaceutical production, and soldiers; andcell aging. Since NF-κB is constitutively active in many tumor cells,the induction of NF-κB activity may protect normal cells from apoptosiswithout providing a beneficial effect to tumor cells. Once the normalcells are repaired, NF-κB activity may be restored to normal levels.NF-κB activity may be induced to protect such radiation- andchemotherapy-sensitive tissues as the hematopoietic system (includingimmune system), the epithelium of the gut, and hair follicles.

Inducers of NF-κB activity may also be used for several otherapplications. Pathological consequences and death caused by exposure ofmammals to a variety of severe conditions including, but not limited to,radiation, wounding, poisoning, infection, aging, and temperature shock,may result from the activity of normal physiological mechanisms ofstress response, such as induction of programmed cell death (apoptosis)or release of bioactive proteins, cytokines.

Apoptosis normally functions to “clean” tissues from wounded andgenetically damaged cells, while cytokines serve to mobilize the defensesystem of the organism against the pathogen. However, under conditionsof severe injury both stress response mechanisms can by themselves actas causes of death. For example, lethality from radiation may resultfrom massive p53-mediated apoptosis occurring in hematopoietic, immuneand digestive systems. Rational pharmacological regulation of NF-κB mayincrease survival under conditions of severe stress. Control over thesefactors may allow control of both inflammatory response and thelife-death decision of cells from the injured organs. Tissues that maybe protected from apoptosis by administering NF-κB inducers include, butare not limited to, the GI tract, lungs, kidneys, liver, cardiovascularsystem, blood vessel endothelium, central and peripheral neural system,hematopoietic progenitor cells, immune system, and hair follicles.

The protective role of NF-κB is mediated by transcriptional activationof multiple genes coding for: a) anti-apoptotic proteins that block bothmajor apoptotic pathways, b) cytokines and growth factors that induceproliferation and survival of HP and other stem cells, and c) potentROS-scavenging antioxidant proteins, such as MnSOD (SOD-2). Thus, bytemporal activation of NF-κB for radioprotection, it may be possible toachieve not only suppression of apoptosis in cancer patients, but alsothe ability to reduce the rate of secondary cancer incidence because ofsimultaneous immunostimulatory effect, which, may be achieved ifactivation of NF-κB is reached via activation of Toll-like receptors.

Another attractive property of the NF-κB pathway as a target is itsactivation by numerous natural factors that can be considered ascandidate radioprotectants. Among these, are multiplepathogen-associated molecular patterns (PAMPs). PAMPs are molecules thatare not found in the host organism, are characteristic for large groupsof pathogens, and cannot be easily mutated. They are recognized byToll-like receptors (TLRs), the key sensor elements of innate immunity.TLRs act as a first warning mechanism of immune system by inducingmigration and activation of immune cells directly or through cytokinerelease. TLRs are type I membrane proteins, known to work as homo- andheterodimers. Upon ligand binding, TLRs recruit MyD88 protein, anindispensable signaling adaptor for most TLRs. The signaling cascadethat follows leads to effects including (i) activation of NF-κB pathway,and (ii) activation of MAPKs, including Jun N-terminal kinease (JNK).The activation of the NF-κB pathway by Toll-like receptor ligands makesthe ligands attractive as potential radioprotectors. Unlike cytokines,many PAMPs have little effect besides activating TLRs and thus areunlikely to produce side effects. Moreover, many PAMPs are present inhumans.

Consistently with their function of immunocyte activation, all TLRs areexpressed in spleen and peripheral blood leukocytes, with moreTLR-specific patterns of expression in other lymphoid organs and subsetsof leukocytes. However, TLRs are also expressed in other tissues andorgans of the body, e.g., TLR1 is expressed ubiquitously, TLR5 is alsofound in GI epithelium and endothelium, while TLRs 2, 6, 7 and 8 areknown to be expressed in lung.

1. Definitions

It is to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting. It must be noted that, as used in the specification and theappended claims, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise.

As used herein, the terms “administer” when used to describe the dosageof an agent that induces NF-κB activity, means a single dose or multipledoses of the agent.

As used herein, the term “analog”, when used in the context of a peptideor polypeptide, means a peptide or polypeptide comprising one or morenon-standard amino acids or other structural variations from theconventional set of amino acids.

As used herein, the term “antibody” means an antibody of classes IgG,IgM, IgA, IgD or IgE, or fragments, fragments or derivatives thereof,including Fab, F(ab′)₂, Fd, and single chain antibodies, diabodies,bispecific antibodies, bifunctional antibodies and derivatives thereof.The antibody may be a monoclonal antibody, polyclonal antibody, affinitypurified antibody, or mixtures thereof which exhibits sufficient bindingspecificity to a desired epitope or a sequence derived therefrom. Theantibody may also be a chimeric antibody. The antibody may bederivatized by the attachment of one or more chemical, peptide, orpolypeptide moieties known in the art. The antibody may be conjugatedwith a chemical moiety.

As used herein, “apoptosis” refers to a form of cell death that includesprogressive contraction of cell volume with the preservation of theintegrity of cytoplasmic organelles; condensation of chromatin (i.e.,nuclear condensation), as viewed by light or electron microscopy; and/orDNA cleavage into nucleosome-sized fragments, as determined bycentrifuged sedimentation assays. Cell death occurs when the membraneintegrity of the cell is lost (e.g., membrane blebbing) with engulfmentof intact cell fragments (“apoptotic bodies”) by phagocytic cells.

As used herein, the term “cancer” means any malignant growth or tumorcaused by abnormal and uncontrolled cell division; it may spread toother parts of the body through the lymphatic system or the bloodstream.

As used herein, the term “cancer treatment” means any treatment forcancer known in the art including, but not limited to, chemotherapy andradiation therapy.

As used herein, the term “combination with” when used to describeadministration of an agent that induces NF-κB activity and an additionaltreatment means that the agent may be administered prior to, togetherwith, after, or metronomically with the additional treatment. The term“together with,” “simultaneous” or “simultaneously” as used herein,means that the additional treatment and the agent of this invention areadministered within 48 hours, preferably 24 hours, more preferably 12hours, yet more preferably 6 hours, and most preferably 3 hours or less,of each other. The term “metronomically” as used herein means theadministration of the agent at times different from the additionaltreatment and at certain frequency relative to repeat administrationand/or the additional treatment.

The agent may be administered at any point prior to the additionaltreatment including, but not limited to, about 48 hr, 46 hr, 44 hr, 42hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30 hr, 28 hr, 26 hr, 24 hr, 22hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10 hr, 8 hr, 6 hr, 4 hr, 3 hr, 2hr, or 1 hr prior to the additional treatment. The agent may beadministered at any point after the additional treatment including, butnot limited to, about 1 hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr,14 hr, 16 hr, 18 hr, 20 hr, 22 hr, 24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34hr, 36 hr, 38 hr, 40 hr, 42 hr, 44 hr, 46 hr, or 48 hr after exposure.

As used herein, the term “derivative”, when used in the context of apeptide or polypeptide, means a peptide or polypeptide different otherthan in primary structure (amino acids and amino acid analogs). By wayof illustration, derivatives may differ by being glycosylated, one formof post-translational modification. For example, peptides orpolypeptides may exhibit glycosylation patterns due to expression inheterologous systems. If at least one biological activity is retained,then these peptides or polypeptides are derivatives according to theinvention. Other derivatives include, but are not limited to, fusionpeptides or fusion polypeptides having a covalently modified N- orC-terminus, PEGylated peptides or polypeptides, peptides or polypeptidesassociated with lipid moieties, alkylated peptides or polypeptides,peptides or polypeptides linked via an amino acid side-chain functionalgroup to other peptides, polypeptides or chemicals, and additionalmodifications as would be understood in the art.

As used herein, the term “fragment”, when used in the context of apeptide or polypeptide, means a portion of a reference peptide orpolypeptide.

As used herein, the term “homolog”, when used in the context of apeptide or polypeptide, means a peptide or polypeptide sharing a commonevolutionary ancestor.

As used herein, the term “treat” or “treating” when referring toprotection of a mammal from a condition, means preventing, suppressing,repressing, or eliminating the condition. Preventing the conditioninvolves administering a composition of this invention to a mammal priorto onset of the condition. Suppressing the condition involvesadministering a composition of this invention to a mammal afterinduction of the condition but before its clinical appearance.Repressing the condition involves administering a composition of thisinvention to a mammal after clinical appearance of the condition suchthat the condition is reduced or maintained. Elimination the conditioninvolves administering a composition of this invention to a mammal afterclinical appearance of the condition such that the mammal no longersuffers the condition.

As used herein, the term “tumor cell” means any cell associated with acancer.

As used herein, the term “variant”, when used in the context of apeptide or polypeptide, means a peptide or polypeptide that differs inamino acid sequence by the insertion, deletion, or conservativesubstitution of amino acids, but retain at least one biologicalactivity. Representative examples of “biological activity” include, butare not limited to, the ability to bind to TLR5 and to be bound by aspecific antibody. A conservative substitution of an amino acid, i.e.,replacing an amino acid with a different amino acid of similarproperties (e.g., hydrophilicity, degree and distribution of chargedregions) is recognized in the art as typically involving a minor change.These minor changes can be identified, in part, by considering thehydropathic index of amino acids, as understood in the art. Kyte et al.,J. Mol. Biol. 157:105-132 (1982). The hydropathic index of an amino acidis based on a consideration of its hydrophobicity and charge. It isknown in the art that amino acids of similar hydropathic indexes can besubstituted and still retain protein function. In one aspect, aminoacids having hydropathic indexes of ±2 are substituted. Thehydrophilicity of amino acids can also be used to reveal substitutionsthat would result in proteins retaining biological function. Aconsideration of the hydrophilicity of amino acids in the context of apeptide permits calculation of the greatest local average hydrophilicityof that peptide, a useful measure that has been reported to correlatewell with antigenicity and immunogenicity. U.S. Pat. No. 4,554,101,incorporated herein by reference. Substitution of amino acids havingsimilar hydrophilicity values can result in peptides retainingbiological activity, for example immunogenicity, as is understood in theart. In one aspect, substitutions are performed with amino acids havinghydrophilicity values within ±2 of each other. Both the hyrophobicityindex and the hydrophilicity value of amino acids are influenced by theparticular side chain of that amino acid. Consistent with thatobservation, amino acid substitutions that are compatible withbiological function are understood to depend on the relative similarityof the amino acids, and particularly the side chains of those aminoacids, as revealed by the hydrophobicity, hydrophilicity, charge, size,and other properties.

2. Methods of Treatment

a. Constitutively Active NF-κB Tumor

This invention relates to a method of treating a mammal suffering from aconstitutively active NF-κB cancer comprising administering to themammal a composition comprising a therapeutically effective amount of anagent that induces NF-κB activity. The agent that induces NF-κB activitymay be administered in combination with a cancer treatment, such aschemotherapy and radiation therapy.

The cancer treatment may comprise administration of a cytotoxic agent orcytostatic agent, or combination thereof. Cytotoxic agents preventcancer cells from multiplying by: (1) interfering with the cell'sability to replicate DNA and (2) inducing cell death and/or apoptosis inthe cancer cells. Cytostatic agents act via modulating, interfering orinhibiting the processes of cellular signal transduction which regulatecell proliferation and sometimes at low continuous levels.

Classes of compounds that may be used as cytotoxic agents include, butare not limited to, the following: alkylating agents (including, withoutlimitation, nitrogen mustards, ethylenimine derivatives, alkylsulfonates, nitrosoureas and triazenes): uracil mustard, chlormethine,cyclophosphamide (Cytoxan®), ifosfamide, melphalan, chlorambucil,pipobroman, triethylene-melamine, triethylenethiophosphoramine,busulfan, carmustine, lomustine, streptozocin, dacarbazine, andtemozolomide; antimetabolites (including, without limitation, folic acidantagonists, pyrimidine analogs, purine analogs and adenosine deaminaseinhibitors): methotrexate, 5-fluorouracil, floxuridine, cytarabine,6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatine,and gemcitabine; natural products and their derivatives (for example,vinca alkaloids, antitumor antibiotics, enzymes, lymphokines andepipodophyllotoxins): vinblastine, vincristine, vindesine, bleomycin,dactinomycin, daunorubicin, doxorubicin, epirubicin, idarubicin, ara-c,paclitaxel (paclitaxel is commercially available as Taxol®),mithramycin, deoxyco-formycin, mitomycin-c, 1-asparaginase, interferons(preferably IFN-α), etoposide, and teniposide. Other proliferativecytotoxic agents are navelbene, CPT-11, anastrazole, letrazole,capecitabine, reloxafine, cyclophosphamide, ifosamide, and droloxafine.

Microtubule affecting agents interfere with cellular mitosis and arewell known in the art for their cytotoxic activity. Microtubuleaffecting agents useful in the invention include, but are not limitedto, allocolchicine (NSC 406042), halichondrin B (NSC 609395), colchicine(NSC 757), colchicine derivatives (e.g., NSC 33410), dolastatin 10 (NSC376128), maytansine (NSC 153858), rhizoxin (NSC 332598), paclitaxel(Taxol®, NSC 125973), Taxol® derivatives (e.g., derivatives (e.g., NSC608832), thiocolchicine (NSC 361792), trityl cysteine (NSC 83265),vinblastine sulfate (NSC 49842), vincristine sulfate (NSC 67574),natural and synthetic epothilones including but not limited toepothilone A, epothilone B, and discodermolide (see Service, (1996)Science, 274:2009) estramustine, nocodazole, MAP4, and the like.Examples of such agents are also described in Bulinski (1997) J. CellSci. 110:3055 3064; Panda (1997) Proc. Natl. Acad. Sci. USA94:10560-10564; Muhlradt (1997) Cancer Res. 57:3344-3346; Nicolaou(1997) Nature 387:268-272; Vasquez (1997) Mol. Biol. Cell. 8:973-985;and Panda (1996) J. Biol. Chem 271:29807-29812.

Also suitable are cytotoxic agents such as epidophyllotoxin; anantineoplastic enzyme; a topoisomerase inhibitor; procarbazine;mitoxantrone; platinum coordination complexes such as cis-platin andcarboplatin; biological response modifiers; growth inhibitors;antihormonal therapeutic agents; leucovorin; tegafur; and haematopoieticgrowth factors.

Cytostatic agents that may be used include, but are not limited to,hormones and steroids (including synthetic analogs):17α-ethinylestradiol, diethylstilbestrol, testosterone, prednisone,fluoxymesterone, dromostanolone propionate, testolactone,megestrolacetate, methylprednisolone, methyl-testosterone, prednisolone,triamcinolone, hlorotrianisene, hydroxyprogesterone, aminoglutethimide,estramustine, medroxyprogesteroneacetate, leuprolide, flutamide,toremifene, zoladex.

Other cytostatic agents are antiangiogenics such as matrixmetalloproteinase inhibitors, and other VEGF inhibitors, such asanti-VEGF antibodies and small molecules such as ZD6474 and SU6668 arealso included. Anti-Her2 antibodies from Genetech may also be utilized.A suitable EGFR inhibitor is EKB-569 (an irreversible inhibitor). Alsoincluded are Imclone antibody C225 immunospecific for the EGFR, and srcinhibitors.

Also suitable for use as an cytostatic agent is Casodex® (bicalutamide,Astra Zeneca) which renders androgen-dependent carcinomasnon-proliferative. Yet another example of a cytostatic agent is theantiestrogen Tamoxifen® which inhibits the proliferation or growth ofestrogen dependent breast cancer. Inhibitors of the transduction ofcellular proliferative signals are cytostatic agents. Representativeexamples include epidermal growth factor inhibitors, Her-2 inhibitors,MEK-1 kinase inhibitors, MAPK kinase inhibitors, PI3 inhibitors, Srckinase inhibitors, and PDGF inhibitors.

A variety of cancers may be treated according to this inventionincluding, but not limited to, the following: carcinoma including thatof the bladder (including accelerated and metastatic bladder cancer),breast, colon (including colorectal cancer), kidney, liver, lung(including small and non-small cell lung cancer and lungadenocarcinoma), ovary, prostate, testes, genitourinary tract, lymphaticsystem, rectum, larynx, pancreas (including exocrine pancreaticcarcinoma), esophagus, stomach, gall bladder, cervix, thyroid, and skin(including squamous cell carcinoma); hematopoietic tumors of lymphoidlineage including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkinslymphoma, non-Hodgkins lymphoma, hairy cell lymphoma, histiocyticlymphoma, and Burketts lymphoma; hematopoietic tumors of myeloid lineageincluding acute and chronic myelogenous leukemias, myelodysplasticsyndrome, myeloid leukemia, and promyelocytic leukemia; tumors of thecentral and peripheral nervous system including astrocytoma,neuroblastoma, glioma, and schwannomas; tumors of mesenchymal originincluding fibrosarcoma, rhabdomyoscarcoma, and osteosarcoma; and othertumors including melanoma, xenoderma pigmentosum, keratoactanthoma,seminoma, thyroid follicular cancer, teratocarcinoma, and cancers of thegastrointestinal tract or the abdominopelvic cavity.

b. Treatment of Side Effects from Cancer Treatment This invention alsorelates to a method of treating a mammal suffering from damage to normaltissue attributable to treatment of a constitutively active NF-κBcancer, comprising administering to the mammal a composition comprisinga therapeutically effective amount of an agent that induces NF-κBactivity. The agent that induces NF-κB activity may be administered incombination with a cancer treatment described above.

c. Modulation of Cell Aging

This invention also relates to a method of modulating cell aging in amammal, comprising administering to the mammal a therapeuticallyeffective amount of an agent that induces NF-κB activity. The agent thatinduces NF-κB activity may be administered in combination with othertreatments.

d. Treatment of Stress

This invention also relates to a method of treating a mammal sufferingfrom damage to normal tissue attributable to stress, comprisingadministering to the mammal a composition comprising a therapeuticallyeffective amount of an agent that induces NF-κB activity. The agent thatinduces NF-κB activity may be administered in combination with othertreatments. The stress may be attributable to any source including, butnot limited to, radiation, wounding, poisoning, infection, andtemperature shock.

e. Radiation

This invention is also related to the protection of cells from theeffects of exposure to radiation. Injury and death of normal cells fromionizing radiation is a combination of direct radiation-induced damageto the exposed cells and an active genetically programmed cell reactionto radiation-induced stress resulting in suicidal death or apoptosis.Apoptosis plays a key role in massive cell loss occurring in severalradiosensitive organs (i.e., hematopoietic and immune systems,epithelium of digestive tract, etc.), the failure of which determinesgeneral radiosensitivity of the organism.

Exposure to ionizing radiation (IR) may be short- or long-term, it maybe applied as a single or multiple doses, to the whole body or locally.Thus, nuclear accidents or military attacks may involve exposure to asingle high dose of whole body irradiation (sometimes followed by along-term poisoning with radioactive isotopes). The same is true (withstrict control of the applied dose) for pretreatment of patients forbone marrow transplantation when it is necessary to preparehematopoietic organs for donor's bone marrow by “cleaning” them from thehost blood precursors. Cancer treatment may involve multiple doses oflocal irradiation that greatly exceeds lethal dose if it were applied asa total body irradiation. Poisoning or treatment with radioactiveisotopes results in a long-term local exposure to radiation of targetedorgans (e.g., thyroid gland in the case of inhalation of 125I). Finally,there are many physical forms of ionizing radiation differingsignificantly in the severity of biological effects.

At the molecular and cellular level, radiation particles are able toproduce breakage and cross-linking in the DNA, proteins, cell membranesand other macromolecular structures. Ionizing radiation also induces thesecondary damage to the cellular components by giving rise to the freeradicals and reactive oxygen species (ROS). Multiple repair systemscounteract this damage, such as several DNA repair pathways that restorethe integrity and fidelity of the DNA, and antioxidant chemicals andenzymes that scavenge the free radicals and ROS and reduce the oxidizedproteins and lipids. Cellular checkpoint systems detect the DNA defectsand delay cell cycle progression until damage is repaired or decision tocommit cell to growth arrest or programmed cell death (apoptosis) isreached

Radiation can cause damage to mammalian organism ranging from mildmutagenic and carcinogenic effects of low doses to almost instantkilling by high doses. Overall radiosensitivity of the organism isdetermined by pathological alterations developed in several sensitivetissues that include hematopoietic system, reproductive system anddifferent epithelia with high rate of cell turnover.

The acute pathological outcome of gamma irradiation leading to death isdifferent for different doses and is determined by the failure ofcertain organs that define the threshold of the organism's sensitivityto each particular dose. Thus, lethality at lower doses occurs from bonemarrow aplasia, while moderate doses kill faster by inducinggastrointestinal (GI) syndrome. Very high doses of radiation can causealmost instant death eliciting neuronal degeneration.

Organisms that survive a period of acute toxicity of radiation cansuffer from long-term remote consequences that include radiation-inducedcarcinogenesis and fibrosis developing in exposed organs (e.g., kidney,liver or lungs) months and years after irradiation.

Cellular DNA is the major target of IR causing a variety of types of DNAdamage (genotoxic stress) by direct and indirect (free radical-based)mechanisms. All organisms maintain DNA repair system capable ofeffective recovery of radiation-damaged DNA; however, errors in the DNArepair process may lead to mutations.

Tumors are generally more sensitive to gamma radiation and can betreated with multiple local doses that cause relatively low damage tonormal tissue. Nevertheless, in some instances, damage of normal tissuesis a limiting factor in application of gamma radiation for cancertreatment. The use of gamma-irradiation during cancer therapy byconventional, three-dimensional conformal or even more focused BeamCathdelivery has also dose-limiting toxicities caused by cumulative effectof irradiation and inducing the damage of the stem cells of rapidlyrenewing normal tissues, such as bone marrow and gastrointestinal (GI)tract.

At high doses, radiation-induced lethality is associated with so-calledhematopoietic and gastrointestinal radiation syndromes. Hematopoieticsyndrome is characterized by loss of hematopoietic cells and theirprogenitors making it impossible to regenerate blood and lymphoidsystem. The death usually occurs as a consequence of infection (resultof immunosuppression), hemorrhage and/or anemia. GI syndrome is causedby massive cell death in the intestinal epithelium, predominantly in thesmall intestine, followed by disintegration of intestinal wall and deathfrom bacteriemia and sepsis. Hematopoietic syndrome usually prevails atthe lower doses of radiation and leads to a more delayed death than GIsyndrome.

In the past, radioprotectants were typically antioxidants—both syntheticand natural. More recently, cytokines and growth factors have been addedto the list of radioprotectants. The mechanism of their radioprotectionis considered to be a result of a facilitating effect on regeneration ofsensitive tissues. There is no clear functional distinction between bothgroups of radioprotectants, however, since some cytokines induce theexpression of cellular antioxidant proteins, such as manganesesuperoxide dismutase (MnSOD) and metallothionein.

The measure of protection for a particular agent is expressed by dosemodification factor (DMF or DRF). DMF is determined by irradiating theradioprotector treated subject and untreated control subjects with arange of radiation doses and then comparing the survival or some otherendpoints. DMF is commonly calculated for 30-day survival (LD50/30drug-treated divided by LD50/30 vehicle-treated) and quantifies theprotection of the hematopoietic system. In order to estimategastrointestinal system protection, LD50 and DMF are calculated for 6-or 7-day survival. DMF values provided herein are 30-day unlessindicated otherwise.

As shown below, inducers of NF-κB possess strong pro-survival activityat the cellular level and on the organism as a whole. In response tosuper-lethal doses of radiation, inducers of NF-κB inhibit bothgastrointestinal and hematopoietic syndromes, which are the major causesof death from acute radiation exposure. As a result of these properties,inducers of NF-κB may be used to treat the effects of natural radiationevents and nuclear accidents. Moreover, since inducers of NF-κB actsthrough mechanisms different from all presently known radioprotectants,they can be used in combination with other radioprotectants, thereby,dramatically increasing the scale of protection from ionizing radiation.

As opposed to conventional radioprotective agents (e.g., scavengers offree radicals), inducers of NF-κB activity may not reduce primaryradiation-mediated damage but may act against secondary events involvingactive cell reaction to primary damage, therefore complementing theexisting lines of defense. Pifithrin-alpha, a pharmacological inhibitorof p53 (a key mediator of radiation response in mammalian cells), is anexample of this new class of radioprotectants. However, the activity ofp53 inhibitors is limited to protection of the hematopoietic system andhas no protective effect in digestive tract (gastrointestinal syndrome),therefore, reducing therapeutic value of these compounds. Anti-apoptoticpharmaceuticals with broader range of activity are desperately needed.

Inducers of NF-κB may be used as a radioprotective agent to extend therange of tolerable radiation doses by increasing radioresistance beyondthe levels achievable by currently available measures (shielding andapplication of existing bioprotective agents) and drastically increasethe chances of survival, for example, in case of onboard nuclearaccidents or large-scale solar particle events. With an approximate DMF(30-day survival) greater than 1.5, the NF-κB inducer flagellin is moreeffective than any currently reported natural compound.

Inducers of NF-κB may be also useful for treating irreplaceable cellloss caused by low-dose irradiation, for example, in the central nervoussystem and reproductive organs. Inducers of NF-κB may also be usedduring cancer chemotherapy to treat the side effects associated withchemotherapy, including alopecia.

In one embodiment, a mammal is treated for exposure to radiation,comprising administering to the mammal a composition comprising atherapeutically effective amount of a composition comprising an inducerof NF-κB. The composition comprising an inducer of NF-κB may beadministered in combination with one or more radioprotectants. The oneor more radioprotectants may be any agent that treats the effects ofradiation exposure including, but not limited to, antioxidants, freeradical scavengers and cytokines.

Inducers of NF-κB may inhibit radiation-induced programmed cell death inresponse to damage in DNA and other cellular structures; however,inducers of NF-κB may not deal with damage at the cellular level and maynot prevent mutations. Free radicals and reactive oxygen species (ROS)are the major cause of mutations and other intracellular damage.Antioxidants and free radical scavengers are effective at preventingdamage by free radicals. The combination of an inducer of NF-κB and anantioxidant or free radical scavenger may result in less extensiveinjury, higher survival, and improved health for exposure. Antioxidantsand free radical scavengers that may be used in the practice of theinvention include, but are not limited to, thiols, such as cysteine,cysteamine, glutathione and bilirubin; amifostine (WR-2721); vitamin A;vitamin C; vitamin E; and flavonoids such as orientin and viceninderived from Indian holy basil (Ocimum sanctum).

Inducers of NF-κB may also be administered in combination with a numberof cytokines and growth factors that confer radioprotection byreplenishing and/or protecting the radiosensitive stem cell populations.Radioprotection with minimal side effects may be achieved by the use ofstem cell factor (SCF, c-kit ligand), Flt-3 ligand, and interleukin-1fragment IL-1b-rd. Protection may be achieved through induction ofproliferation of stem cells (all mentioned cytokines), and prevention oftheir apoptosis (SCF). The treatment allows accumulation of leukocytesand their precursors prior to irradiation thus enabling quickerreconstitution of the immune system after irradiation. SCF efficientlyrescues lethally irradiated mice with DMF in the range of 1.3-1.35 andis also effective against gastrointestinal syndrome. Flt-3 ligand alsoprovides strong protection in mice (70-80% 30-day survival at LD100/30,equivalent to DMF>1.2) and rabbits.

In addition, combinations of cytokines may provide enhancedradioprotection, such as: TPO combined with interleukin 4 (IL-4) and/orinterleukin 11 (IL-11); GM-CSF combined with IL-3; G-CSF combined withFlt-3 ligand; 4F combination: SCF, Flt-3 ligand, TPO and IL-3; and 5Fcombination: 4F with addition of SDF-1.

In addition, gastrointestinal radioprotectors may be used, includingtransforming growth factor beta3 (TGFb3), interleukin 11 (IL-11), andmentioned keratinocyte growth factor (KGF). While these radioprotectorsalso protect the intestine, they are likely to synergize with flagellinor flagellin related polypeptides since the results below show thatflagellin and flagellin related polypeptides protect endothelium, whilethese gastrointestinal radioprotectors protect epithelium of GI tract.

Several factors, while not cytokines by nature, stimulate theproliferation of immunocytes and may be used in combination withinducers of NF-κB. For example, 5-AED (5-androstenediol) is a steroidthat stimulates the expression of cytokines and increases resistance tobacterial and viral infections. A subcutaneous injection of 5-AED inmice 24 h before irradiation improved survival with DMF=1.26. Syntheticcompounds, such as ammonium tri-chloro(dioxoethylene-O,O′—) tellurate(AS-101), may also be used to induce secretion of numerous cytokines andfor combination with inducers of NF-κB. Additional radioprotectorsinclude, growth hormone (GH), thrombopoietin (TPO), interleukin 3(IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF),granulocyte colony-stimulating factor (G-CSF), and stromal derivedfactor-1 (SDF-1).

Growth factors and cytokines may also be used to provide protectionagainst gastrointestinal syndrome. Keratinocyte growth factor (KGF)promotes proliferation and differentiation in the intestinal mucosa, andincreases the post-irradiation cell survival in the intestinal crypts.Hematopoietic cytokine and radioprotectant SCF may also increaseintestinal stem cell survival and associated short-term organismsurvival.

Inducers of NF-κB may offer protection against both gastrointestinal(GI) and hematopoietic syndromes. Since mice exposed to 15 Gy ofwhole-body lethal irradiation die mostly from GI syndrome, a compositioncomprising an inducer of NF-κB and one or more inhibitors of GI syndromemay be more effective. Inhibitors of GI syndrome that may be used in thepractice of the invention include, but are not limited to, cytokinessuch as SCF and KGF.

The composition comprising an inducer of NF-κB may be administered atany point prior to exposure to radiation including, but not limited to,about 48 hr, 46 hr, 44 hr, 42 hr, 40 hr, 38 hr, 36 hr, 34 hr, 32 hr, 30hr, 28 hr, 26 hr, 24 hr, 22 hr, 20 hr, 18 hr, 16 hr, 14 hr, 12 hr, 10hr, 8 hr, 6 hr, 4 hr, 3 hr, 2 hr, or 1 hr prior to exposure. Thecomposition comprising an inducer of NF-κB may be administered at anypoint after exposure to radiation including, but not limited to, about 1hr, 2 hr, 3 hr, 4 hr, 6 hr, 8 hr, 10 hr, 12 hr, 14 hr, 16 hr, 18 hr, 20hr, 22 hr, 24 hr, 26 hr, 28 hr, 30 hr, 32 hr, 34 hr, 36 hr, 38 hr, 40hr, 42 hr, 44 hr, 46 hr, or 48 hr after exposure to radiation.

f. Sepsis

This invention also relates to a method of preventing sepsis in a mammalcomprising administering to the mammal a composition comprising atherapeutically effective amount of an agent that induces NF-κBactivity. The agent that induces NF-κB activity may be administered incombination with other treatments.

Viral or bacterial infections may stimulate the innate immune systemthrough Toll-like receptor (TLR) ligands. Macrophages may be protectedand/or stimulated by flagellin and flagellin related polypeptides due tothe presence of TLR5 on their surface. For example, a crucial step inthe development of an anthrax infection is death of macrophages killedfrom within by B. anthracis. Protection of intestinal endotheliumagainst various stresses using flagellin and flagellin relatedpolypeptides may prevent GI cell death and also may prevent penetrationof the GI wall by infectious agent, thereby preventing GI bleedingcaused by infections such as Ebola. Other hemorrhagic viral infectionsmay also be prevented by rescue of endothelium and gastrointestinalepithelium.

3. Agent

This invention also relates to an agent that induces NF-κB activity. Theagent may be an artificially synthesized compound or a naturallyoccurring compound. The agent may be a low molecular weight compound,polypeptide or peptide, or a fragment, analog, homolog, variant orderivative thereof.

The agent may also be an NF-κB inducing cytokine including, but notlimited to, IL2, IL6, TNF and TGFβ. The agent may also be aprostaglandin. The agent may also be a growth factor including, but notlimited to, KGF and PDGF. The agent may also be an antibody that inducesNF-κB activity.

a. Flagellin

In one embodiment, the agent that induces NF-κB activity is flagellin.As shown in the Examples below, flagellin and flagellin relatedpolypeptides possess strong pro-survival activity at the cellular leveland for the organism as a whole. Interestingly, flagellin alsostimulates natural killer (NK) cells and T-lymphocytes, which are themajor components of anti-tumor immunity (Tsujimoto H, et. al., J LeukocBiol. 2005 October; 78(4):888-97; Caron G., et. al., J Immunol. 2005Aug. 1; 175(3):1551-7; Honko A N & Mizel S B, Immunol Res. 2005;33(1):83-101). As a result, flagellin may be used as a radioprotectantin cancer treatments.

The present invention is also related to flagellin related polypeptides,such as those polypetpides described herein. As used herein, the term“flagellin” is intended to mean a flagellin or flagellin-relatedpolypeptide from any source, including a variety of Gram-positive andGram-negative bacterial species. The amino acid sequences of flagellinfrom 23 bacterial species are depicted in FIG. 7 of U.S. PatentPublication No. 2003/0044429, the contents of which are incorporatedherein by reference. The nucleotide sequences encoding the flagellinpolypeptides listed in FIG. 7 of U.S. 2003/0044429 are publicallyavailable at sources including the NCBI Genbank database.

Flagellin is the major component of bacterial flagellum. Flagellin iscomposed of three domains (FIG. 9). Domain 1 (D1) and domain 2 (D2) arediscontinuous and are formed when residues in the amino terminus andcarboxy terminus are juxtaposed by the formation of a hairpin structure.The amino and carboxy terminus comprising the D1 and D2 domains is mostconserved, whereas the middle hypervariable domain (D3) is highlyvariable. Studies with a recombinant protein containing the amino D1 andD2 and carboxyl D1 and D2 separated by an Escherichia coli hinge(ND1-2/ECH/CD2) indicate that D1 and D2 are bioactive when coupled to anECH element. This chimera, but not the hinge alone, induced I_(κ)B_(α)degradation, NF-κB activation, and NO and IL-8 production in twointestinal epithelial cell lines. The non-conserved D3 domain is on thesurface of the flagellar filament and contains the major antigenicepitopes. The potent proinflammatory activity of flagellin may reside inthe highly conserved N and C D1 and D2 regions.

Flagellin induces NF-κB activity by binding to Toll-like receptor 5(TLR5). The TLR family is composed of at least 10 members and isessential in innate immune defense against pathogens. The innate immunesystem recognizes pathogen-associated molecular patterns (PAMPs) thatare conserved on microbial pathogens. TLR may recognize a conservedstructure that is particular to bacterial flagellin. The conservedstructure may be comprised of a large group of residues that aresomewhat permissive to variation in amino acid content. Smith et al.,Nat Immunol. 4:1247-53 (2003) have identified 13 conserved amino acidsin flagellin that are part of the conserved structure recognized byTLR5. The 13 conserved amino acids of flagellin important for TLR5activity are shown in FIG. 24.

In a preferred embodiment, the flagellin is from a species ofSalmonella, a representative example of which is S. dublin (encoded byGenBank Accession Number M84972) (SEQ ID NO: 1). In another preferredembodiment, the flagellin related-polypeptide is a fragment, variant,analog, homolog, or derivative of SEQ ID NO: 1, or combination thereof,that binds to TLR5 and induces TLR5-mediated activity, such asactivation of NF-κB activity. A fragment, variant, analog, homolog, orderivative of flagellin may be obtained by rational-based design basedon the domain structure of Flagellin and the conserved structurerecognized by TLR5.

In a more preferred embodiment, the fragment, variant, analog, homolog,or derivative of SEQ ID NO: 1, or combination thereof, comprises atleast 10, 11, 12, or 13 of the 13 conserved amino acids shown in FIG. 24(positions 89, 90, 91, 95, 98, 101, 115, 422, 423, 426, 431, 436 and452). In another more preferred embodiment, the amino- andcarboxy-terminus of the fragment, variant, analog, homolog, orderivative of SEQ ID NO: 1, or combination thereof, is at least 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,98%, or 99% identical to amino acids 1-174 and 418-505 of SEQ ID NO: 1.FIG. 26 lists the percentage identity of the amino- and carboxy-terminusof flagellin with known TLR-5 stimulating activity, as compared to SEQID NO: 1.

Flagellin homologs may be a flagellin polypeptide from any Gram-positiveor Gram-negative bacterial species including, but not limited to, theflagellin polypeptides disclosed in U.S. Pat. Pub. 2003/000044429, thecontents of which are incorporated herein, and the flagellin peptidescorresponding to the Accession numbers listed in the BLAST results shownin FIG. 25. Also contemplated, are fragments, variants, analogs andderivatives of flagellin homologs.

Flagellin fragments may be portions of a flagellin polypeptide thatstimulate TLR5 activity. Numerous deletional mutants of flagellin havebeen made that retain at least some TLR5 stimulating activity. Inaddition to the deletional mutants disclosed in the Examples herein,representative deletional mutants include translation of GenBankAccession number D13689 missing amino acids 185-306 or 444-492, andtranslation of GenBank Accession number M84973 missing amino acids179-415. Also contemplated, are homologs, variants, analogs andderivatives of flagellin fragments.

Flagellin variants include flagellin polypeptides with transposoninsertions and changes to the variable D3 domain. The D3 domain may besubstituted in part, or in whole, with a hinge or linker polypeptidethat allows the D1 and D2 domains to properly fold such that the variantstimulates TLR5 activity. Representative examples of variant hingeelements may be found in the E. coli MukB protein and SEQ ID NOS: 3 and4. Also contemplated, are fragments, homologs, analogs and derivativesof flagellin variants.

4. Composition

This invention also relates to a composition comprising atherapeutically effective amount of an inducer of NF-κB. The compositionmay be a pharmaceutical composition, which may be produced using methodswell known in the art. As described above, the composition comprising aninducer of NF-κB may be administered to a mammal for the treatment ofconditions associated with apoptosis including, but not limited to,exposure to radiation, side effect from cancer treatments, stress andcell aging. The composition may also comprise additional agentsincluding, but not limited to, a radioprotectant or a chemotherapeuticdrug.

a. Administration

Compositions of this invention may be administered in any mannerincluding, but not limited to, orally, parenterally, sublingually,transdermally, rectally, transmucosally, topically, via inhalation, viabuccal administration, intrapleurally, or combinations thereof.Parenteral administration includes, but is not limited to, intravenous,intraarterial, intraperitoneal, subcutaneous, intramuscular,intrathecal, and intraarticular. Transmucosally administration includes,but is not limited to intranasal. For veterinary use, the compositionmay be administered as a suitably acceptable formulation in accordancewith normal veterinary practice. The veterinarian can readily determinethe dosing regimen and route of administration that is most appropriatefor a particular animal.

The composition may be administered prior to, after or simultaneouslywith a stress that triggers apoptosis, or a combination thereof. Thecomposition may be administered from about 1 hour to about 48 hoursprior to or after exposure to a stress that triggers apoptosis.

b. Formulation

Compositions of this invention may be in the form of tablets or lozengesformulated in a conventional manner. For example, tablets and capsulesfor oral administration may contain conventional excipients including,but not limited to, binding agents, fillers, lubricants, disintegrantsand wetting agents. Binding agents include, but are not limited to,syrup, accacia, gelatin, sorbitol, tragacanth, mucilage of starch andpolyvinylpyrrolidone. Fillers include, but are not limited to, lactose,sugar, microcrystalline cellulose, maizestarch, calcium phosphate, andsorbitol. Lubricants include, but are not limited to, magnesiumstearate, stearic acid, talc, polyethylene glycol, and silica.Disintegrants include, but are not limited to, potato starch and sodiumstarch glycollate. Wetting agents include, but are not limited to,sodium lauryl sulfate). Tablets may be coated according to methods wellknown in the art.

Compositions of this invention may also be liquid formulationsincluding, but not limited to, aqueous or oily suspensions, solutions,emulsions, syrups, and elixirs. The compositions may also be formulatedas a dry product for constitution with water or other suitable vehiclebefore use. Such liquid preparations may contain additives including,but not limited to, suspending agents, emulsifying agents, nonaqueousvehicles and preservatives. Suspending agent include, but are notlimited to, sorbitol syrup, methyl cellulose, glucose/sugar syrup,gelatin, hydroxyethylcellulose, carboxymethyl cellulose, aluminumstearate gel, and hydrogenated edible fats. Emulsifying agents include,but are not limited to, lecithin, sorbitan monooleate, and acacia.Nonaqueous vehicles include, but are not limited to, edible oils, almondoil, fractionated coconut oil, oily esters, propylene glycol, and ethylalcohol. Preservatives include, but are not limited to, methyl or propylp-hydroxybenzoate and sorbic acid.

Compositions of this invention may also be formulated as suppositories,which may contain suppository bases including, but not limited to, cocoabutter or glycerides. Compositions of this invention may also beformulated for inhalation, which may be in a form including, but notlimited to, a solution, suspension, or emulsion that may be administeredas a dry powder or in the form of an aerosol using a propellant, such asdichlorodifluoromethane or trichlorofluoromethane. Compositions of thisinvention may also be formulated transdermal formulations comprisingaqueous or nonaqueous vehicles including, but not limited to, creams,ointments, lotions, pastes, medicated plaster, patch, or membrane.

Compositions of this invention may also be formulated for parenteraladministration including, but not limited to, by injection or continuousinfusion. Formulations for injection may be in the form of suspensions,solutions, or emulsions in oily or aqueous vehicles, and may containformulation agents including, but not limited to, suspending,stabilizing, and dispersing agents. The composition may also be providedin a powder form for reconstitution with a suitable vehicle including,but not limited to, sterile, pyrogen-free water.

Compositions of this invention may also be formulated as a depotpreparation, which may be administered by implantation or byintramuscular injection. The compositions may be formulated withsuitable polymeric or hydrophobic materials (as an emulsion in anacceptable oil, for example), ion exchange resins, or as sparinglysoluble derivatives (as a sparingly soluble salt, for example).

c. Dosage

A therapeutically effective amount of the agent required for use intherapy varies with the nature of the condition being treated, thelength of time that induction of NF-κB activity is desired, and the ageand the condition of the patient, and is ultimately determined by theattendant physician. In general, however, doses employed for adult humantreatment typically are in the range of 0.001 mg/kg to about 200 mg/kgper day. The dose may be about 1 μg/kg to about 100 μg/kg per day. Thedesired dose may be conveniently administered in a single dose, or asmultiple doses administered at appropriate intervals, for example astwo, three, four or more subdoses per day. Multiple doses often aredesired, or required, because NF-κB activity in normal cells may bedecreased once the agent is no longer administered.

The dosage of an inducer of NF-κB may be at any dosage including, butnot limited to, about 1 μg/kg, 25 μg/kg, 50 μg/kg, 75 μg/kg, 100 μg/kg,125 μg/kg, 150 μg/kg, 175 μg/kg, 200 μg/kg, 225 μg/kg, 250 μg/kg, 275μg/kg, 300 μg/kg, 325 μg/kg, 350 μg/kg, 375 μg/kg, 400 μg/kg, 425 μg/kg,450 μg/kg, 475 μg/kg, 500 μg/kg, 525 μg/kg, 550 μg/kg, 575 μg/kg, 600μg/kg, 625 μg/kg, 650 μg/kg, 675 μg/kg, 700 μg/kg, 725 μg/kg, 750 μg/kg,775 μg/kg, 800 μg/kg, 825 μg/kg, 850 μg/kg, 875 μg/kg, 900 μg/kg, 925μg/kg, 950 μg/kg, 975 μg/kg or 1 mg/kg.

This invention has multiple aspects, illustrated by the followingnon-limiting examples.

Example 1 P53 Deficiency Accelerated Development of GI Syndrome in Mice

The primary cause of death from ionizing radiation (IR) of mammalsdepends on the radiation dose. At doses of up to 9-10 Gy, mice die 12-20days later, primarily from lethal bone marrow depletion-hematopoietic(HP) syndrome. At this dose, irradiated mice can be rescued fromlethality by bone marrow transplantation. Animals that receive>15 Gy diebetween 7-12 days after treatment (before hematopoietic syndrome cankill them) from complications of damage to the smallintestine-gastrointestinal (GI) syndrome. In both cases of HP and GIsyndromes, lethal damage of tissues starts from massive p53-dependentapoptosis. This observation allowed us earlier to suggest that p53 couldbe a determinant of radiation-induced death. Consistently, p53-deficientmice were resistant to doses of radiation that kill through HP syndrome,and lethality of wild type animals receiving 6-11 Gy of gamma radiationcould be reduced by temporary pharmacological inhibition of p53 by thesmall molecule p53 inhibitor pifithrin-alpha (PFT) (Komarov et al 1999).Identification of p53 as a factor sensitizing tissues to genotoxicstress was further strengthened by demonstrating the p53 dependence ofhair loss (alopecia) occurring as a result of experimental chemotherapyor radiation. Hence, based on previous observations, one would expectthat p53 continues to play an important role in development of lethal GIsyndrome after higher doses of IR. Surprisingly, p53-deficiencysensitizes mice to higher doses of IR causing lethal gastro-intestinalsyndrome (FIG. 1). Continuous cell proliferation in the crypts ofp53-deficient epithelium after IR correlates with accelerated death ofdamaged cells of crypt and rapid destruction of villi. p53 prolongssurvival by inducing growth arrest in the crypts of small intestinethereby preserving integrity of the guts (FIG. 2). Thus, proapoptoticfunction of p53 promotes hematopoietic syndrome while its growth arrestfunction delays development of gastro-intestinal syndrome.

The dynamics of cell population in the small intestine have beenanalyzed in great detail. Cell proliferation in the epithelia of the gutis limited to the crypts where stem cells and early proliferatingprogenitors are located. After a couple of cell divisions, alreadydifferentiated descendants of crypt stem cells move up the villi to beshed at the villar tip. In the small intestine of the mouse, the entire“trip” of the cell (the proliferative compartment to the tip of thevillus) normally takes between 3 and 5 days. Although reaction of thesmall intestine to gamma radiation has been well examined at apathomorphological level, it still remains unclear what is the exactcause of GI lethality, including the primary event. Death may occur as adirect consequence of the damage of epithelial crypt cells and followeddenudation of villi leading to fluid and electrolyte imbalance,bacteremia and endotoxemia. Besides inflammation and stromal responses,endothelial dysfunctions seem to be the important factors contributingto lethality. In summary, pharmacological suppression of p53 that wasshown to be so effective as a method of protection from IR-induced HPsyndrome, is useless (if not detrimental) against GI syndrome.Therefore, it is necessary to develop alternative approaches toradioprotection of epithelium of small intestine that will rely onanother mechanism, such as, for example, activation of NF-κB andsubsequent inhibition of cell death.

Example 2 Flagellin Delays Mouse Death Caused by IR-Induced GI Syndrome

Whole body irradiation of mice with 15 Gy gamma radiation caused deathwithin 8 days from GI syndrome providing a conventional model ofradiation induced damage of GI tract. To test whether flagellin wascapable of protecting GI epithelium from IR, we tested the effect ofi.v.-injected flagellin on the dynamics of mouse lethality after 15 Gyof radiation. We used a range of flagellin doses, all of which weresignificantly lower than the highest tolerable dose known fromliterature (300 μg/mouse). Irradiation was done 4 hours post treatment.The results of a representative experiment are shown in FIG. 4. Asexpected, control irradiated mice (that received PBS i.v.) died between5 and 8 days post-treatment. Animals that received flagellin livedsignificantly longer; the extension of animal survival correlated withthe dose of flagellin. Pathomorphological analysis of the smallintestine on day 7 after irradiation revealed dramatic differencesbetween flagellin-treated and control groups (FIG. 5). Intravenous,intraperitoneal and subcutaneous delivery of 0.2 mg/kg of flagellinfollowed by 13 Gy irradiation afforded similar degree of protection,leading to 85-90% 30-day survival of mice (data not shown). While notbeing bound by theory, flagellin may be a radioprotectant due to itsactivation of NF-κB, which presumably acts as an inhibitor of apoptoticdeath.

Example 3 Flagellin Rescues Mice from Lethal IR-Induced HematopoieticSyndrome

We next tested whether flagellin had an effect on mouse IR-induced deathfrom HP syndrome that was experimentally induced by lower radiationdoses (usually up to 11 Gy) that are incapable of causing lethal GItoxicity. The experiments were done similarly to the above-describedones (FIGS. 14 and 15), however, instead of 15Gy, mice received 10Gy,the dose that caused 100% killing in control group by day 13 (FIG. 6).Flagellin-treated group (5 μg/mouse) showed complete protection fromthis dose of IR surprisingly indicating that flagellin-mediatedradioprotection acts not only against GI but also against HP IR-inducedsyndromes.

Example 4 Time Dependence on the Protective Effect of Flagellin

Mice were next administered flagellin at different times prior to 13Gyof gamma irradiation. The results of one of such experiments is shown inFIG. 7. The obtained results show that flagellin is effective as aradioprotectant from 13Gy if injected 1-4 h before treatment.

In order to further estimate the dependence of radioprotective activityof flagellin on the time of treatment, mice were injected at severaltime points relative to the moment of gamma-irradiation. Experimentswere performed essentially as explained above, using intraperitonealinjection of 5 μg/mouse (0.2 mg/kg) of full-length flagellin or, forcontrol mice, 5 μg/mouse (0.2 mg/kg) of bacterial RNA polymerase. Theexperiments were performed using the NIH-Swiss mouse strain. The resultsshow that flagellin provides ˜90% survival after 13 Gy irradiation ifinjected at 1 or 2 hours before treatment (FIG. 7). Only −1 h graph isshown for clarity, however, both timepoints (−1 and −2 h) providesimilar degree and dynamics of survival. The 4 h timepoint showssomewhat lower protection. Flagellin injected 24 hours beforeirradiation had no protective effect against 13 Gy induced death.

Interestingly, administration of flagellin 24 hours before 10 Gygamma-irradiation provided 100% protection. While 13 Gy irradiation inmice primarily induces death from GI syndrome, 10 Gy-induced death ismostly mediated by hematopoietic syndrome. Accordingly, such long-termprotection from 10 Gy irradiation may be mediated by enhancedproliferation or survival of hematopoietic stem cell induced byflagellin and/or long-living secondary cytokines.

Example 5 Determination of LD_(50/30), LD₅₀₇ and DMF for Flagellin

We next obtained an estimate of radiation dose-dependent protection forflagellin. As shown above (FIG. 7), treatment with flagellin wassufficient for 100% protection against 10 Gy gamma-irradiation (thisdose causes death from hematopoietic syndrome) and 90% 30-day survivalat 13 Gy (both hematopoietic and GI syndromes). Experiments wereperformed as described above, using flagellin 5 μg/mouse (0.2 mg/kg),intraperitoneally injected 1 h before irradiation.

At 15 Gy, however, 100% 7-day survival was followed by delayed deathafter 13 days (0% 30-day survival), while control group had fullysuccumbed to GI syndrome by day 7 (FIG. 8). The kinetics of theflagellin-treated group mortality after 15 Gy irradiation is reminiscentof such of control group at 10 Gy, hinting at death caused byhematopoietic syndrome. The results provide an estimate of flagellinLD_(50/30) around 13.5-14 Gy and DMF₃₀ of about 1.75-1.8. This degree ofradioprotection is significantly higher than any reported for a naturalcompound.

Example 6 Rational Design and Cloning of Flagellin Fragments

Salmonella flagellin, encoded by the FliC gene (SEQ ID NO: 2), is astrong activator of pro-survival NF-κB pathway. This is the most likelymechanism of its radioprotective action. Previous studies have shownthat binding of flagellin to Toll-like receptor 5 (TLR5) on the cellsurface is a necessary step that triggers activation of NF-κB. Thedomain structure of Salmonella flagellin is described in sufficientdetail in the literature (FIG. 9). Moreover, previous structural studiesof flagellin-TLR5 complex (FIG. 10) provide the ability to distinguishbetween domains that are essential or dispensable for binding and thusNF-κB activation. Protein minimization may provide reduced immuneresponse after repeated administration of flagellin-relatedpolypeptides. This may be achieved, in part, due to lower immunogenicityof low molecular weight proteins and smaller number of immunogenicepitopes available.

The domains needed for TLR5 binding may be located exclusively in theevolutionary conserved N- and C-terminal domains of bacterialflagellins. The hypervariable domain (amino acids 178-402) does not comeinto close contact with TLR5. As was demonstrated previously,replacement of this domain with a flexible linker peptide did notdisrupt binding to TLR5. In addition, N-terminal and C-terminalcoiled-coil polymerization domains (amino acids 1-55, 456-505) do notbind to TLR5 and likely are dispensable (see modified N and C termini Band B′, respectively, as shown herein). Also, another fragmentN-terminus lacking all domains but major N-terminal α-helix thatactually binds TLR5 (amino acids 56-100) may be sufficient for binding.

Accordingly, three types of N-termini (A, B, C) and two types ofC-termini (A′,B′), connected with a flexible linker (SEQ ID NOS: 3 and4) taken from pGEX-KG cloning vector (SEQ ID NOS: 5 and 6) were combinedinto expression constructs to produce several possible flagellinfragments (Table 1). In addition, constructs representing separateN-termini (A, B, C) and glutathione-S-transferase (GST)-fusions ofC-termini (GST-A′, GST-B′) were prepared. All constructs were cloned inthe pRSETb bacterial expression vector and 6×His-tagged proteins wereproduced and purified for further experiments (FIG. 11).

TABLE 1 Name Structure DNA Protein AA′  (1-177)-Linker-(402-505) SEQ IDNO: 7  SEQ ID NO: 8  AB′  (1-177)-Linker-(402-450) SEQ ID NO: 9  SEQ IDNO: 10 BA′ (56-177)-Linker-(402-505) SEQ ID NO: 11 SEQ ID NO: 12 BB′(56-177)-Linker-(402-450) SEQ ID NO: 13 SEQ ID NO: 14 CA′(56-100)-Linker-(402-505) SEQ ID NO: 15 SEQ ID NO: 16 CB′(56-100)-Linker-(402-450) SEQ ID NO: 17 SEQ ID NO: 18 A  (1-177) SEQ IDNO: 19 SEQ ID NO: 20 B (56-177) SEQ ID NO: 21 SEQ ID NO: 22 C (56-100)SEQ ID NO: 23 SEQ ID NO: 24 GST- GST-Linker-(402-505) SEQ ID NO: 25 SEQID NO: 26 A′ GST- GST-Linker-(402-450) SEQ ID NO: 27 SEQ ID NO: 28 B′

Example 7 Selection of Biologically Active Flagellin Fragments

Since the radioprotective activities of flagellin appear to be NF-κBdependent, we tested the ability of the flagellin fragments to induceNF-κB translocation to the nucleus and binding to its target sites inDNA. This was tested by electrophoretic mobility shift assay (EMSA)using nuclear extracts from flagellin- and fragment-treated A549 lungcancer cells and labeled synthetic NF-κB binding kB oligonucleotide.

Only flagellin itself and fragments AA′, AB′, and BA′ were capable ofinducing NF-κB translocation (FIG. 12). The level of translocation iscomparable for flagellin and fragments AA′, AB′, and BA′. Thehypervariable domain does not appear to be necessary for NF-κBtranslocation, while the presence of at least one polymerization domain,N- or C-terminal, is required. Mixtures of the N- and C-terminalfragments (A+A′, A+B′) were inactive.

While translocation of NF-κB to the nucleus is a crucial step ininduction of NF-κB-regulated inhibitors of apoptosis, it is notsufficient in itself. To directly test the ability of selected fragmentsto induce expression of NF-κB-regulated genes, we performed reporterassay experiments. Flagellin and the AA′, BB′, A′ and B′ fragments wereused for treatment of H116 human colon cancer cells carrying luciferasegene under a NF-κB-responsive promoter. The reporter construct containedthree NF-κB-binding sites from the E-selectin promoter combined with aHsp70 minimal promoter that is routinely used for the detection of NF-κBstatus of cells. Luciferase activity was measured in cell lysates sixhours after addition of flagellin or its truncated fragments into themedium. TNF was used as positive control. The results of arepresentative experiment are shown in FIG. 13 and indicates thatflagellin and fragment AA′ are capable of NF-κB activation, whereasfragments BB′, GST-A′ and GST-B′ are not.

Example 8 Further Optimization of Flagellin Fragments

We further minimized the AA′ flagellin fragment by producing additionalfragments through stepwise removal of peptide fragments from itsN-terminal half (Table 2). Electrophoretic mobility shift assays wereperformed as described above using nuclear extracts from flagellin- andfragment-treated HT29 human colon cancer cells and labeled syntheticNF-κB binding κB oligonucleotide. NF-κB binding activity in HT29 cellswas stimulated with TNFα (10 ng/ml), or flagellin fragments (1 mg/ml)for 15 min. As shown in FIG. 14, fragments AA′n1-170, AA′n54-170,AA′n1-163 and AA′n54-163 each induce NF-κB translocation, with levelscomparable to that of flagellin for AA′n1-170, AA′n54-170 and AA′n1-163.

TABLE 2 Name Structure DNA Protein AA′n54-177 (54-177)-Linker-(402-505)SEQ ID NO: 11 SEQ ID NO: 12 AA′n1-170  (1-170)-Linker-(402-505) SEQ IDNO: 29 SEQ ID NO: 30 AA′n54-170 (54-170)-Linker-(402-505) SEQ ID NO: 31SEQ ID NO: 32 AA′n1-163  (1-163)-Linker-(402-505) SEQ ID NO: 33 SEQ IDNO: 34 AA′n54-163 (54-163)-Linker-(402-505) SEQ ID NO: 35 SEQ ID NO: 36

In order to study the ability of the AA′ fragments to directly activateNF-κB-regulated transcription, we performed reporter assay experimentsas described above for a wide range of concentrations of flagellin,original AA′ and AA′-derived fragments. As shown above, AA′ andAA′n1-170 induce NF-κB-regulated transcription at the level comparableto such of flagellin over the studied range of concentrations (FIG. 15,left). AA′ and AA′n1-170 are more active than flagellin in the very lowconcentration range (FIG. 15, right), possibly due to their reducedmolecular weight. The results with fragment AA′n1-170 show thatAA′-derived flagellin fragments may be made with a portion of theN-terminal domain removed without significant loss of activity and maybe used as effective radioprotectors.

The above experiments (EMSA and reporter activation assay) were repeatedwith flagellin and AA′ fragments subjected to 30 minutes boiling andrenaturation before being applied to cells. The results were comparableto those obtained without boiling (data not shown). This shows that theobserved differences in flagellin fragment activity may not be caused bychanges in protein stability.

Example 9 In Vivo Comparison of Radioprotective Properties of Flagellinand Flagellin Fragments

As shown above, full-length flagellin provides protection from bothhematopoietic and gastrointestinal syndromes. The radioprotectivepotential of flagellin fragments was similarly tested aftergamma-irradiation with 11 Gy (dose that induces hematopoieticsyndrome-associated mortality in mice) or 14 Gy (dose that causes deathfrom GI syndrome). Mice (10 animals per group) were injectedsubcutaneously with 5.0 μg/mouse (0.2 mg/kg) of flagellin or itsfragments, AA′ or BB′, and gamma-irradiated 1 hour later.

The degree of radioprotection displayed by the AA′ fragment is at leastcomparable to full-length flagellin (FIG. 16). Both the AA′ fragment andfull-length flagellin showed 100% 30-day survival for mice irradiatedwith 11 Gy and 14 Gy. Meanwhile, 0% of mice injected with the BB′fragment survived to 30 days. This is expected since the BB′ fragment isincapable of inducing NF-κB in vitro. These results show thatsignificant reduction in the size of flagellin (about 40% removed) maybe achieved without a decrease in the degree of radioprotection. Inaddition, the ability to predict radioprotective potential from resultsof in vitro NF-κB activation is confirmed.

Example 10 Identification of Cellular Targets of Flagellin-MediatedRadioprotection

Tissue samples of intestinal mucosa were taken 5 days after 14 Gyirradiation from mice pretreated with flagellin and control mice.Control animals were treated with 5.0 μg/mouse (0.2 mg/kg) bacterialRNA-polymerase. Pathomorphological analysis of the small intestinereveals reduction of the size of crypts and villi and a number of thecells with condensed apoptotic nuclei in control mouse and near-normalmorphology in the treated mouse (data not shown). Tissue samples (smallintestine and skin from the back) were also obtained from mice treatedwith the AA′ fragment. The results shown in FIG. 17 are areas of typicalmorphology observed over a set of at least 3 mice. After treatment withflagellin and the AA′ fragment, mice demonstrated near-normal intestinalmorphology with preservation of the villi/crypt structure (FIG. 17A).

In addition to purely histological observation of cell death andsurvival, we performed more specialized tests of apoptotic cell death inintestinal tissue using a TUNEL assay, which detectsapoptosis-associated DNA fragmentation. These experiments allowed us todefine with a high degree of probability cellular populations that aredepleted by radiation and rescued by flagellin fragment treatment. Theearliest radiation-induced alterations detectable in the small intestineafter treatment with IR is apoptosis occurring in vascular endothelialcells of villi, which is seen as early as 5 hours post treatment (FIG.17B). This apoptosis, which is believed to be critical forradiosensitivity of the small intestine, was almost completely blockedin the mice pretreated with the AA′ fragment (FIG. 17B, bottom panel).Degeneration of villi and crypts, occurring within the next several dayspost treatment and greatly suppressed in AA′ fragment-treated animals,comes as a consequence of injury of blood vessels. Effective protectionof endothelial cells of the small intestine by the flagellin fragmentmay be due to expression of TLR5 in these cells.

Remarkably, the AA′ fragment and flagellin also prevented theradiation-induced disappearance of sebaceous glands located at the baseof skin hair follicles (FIG. 27C). These results further confirm thesuitability of AA′ fragment for radioprotection and for the preventionof radiation-induced hair loss.

Example 11 Protection from Supralethal Radiation

In order to explore the limits of radioprotection provided by the AA′fragment, we irradiated mice with 17 Gy and 20 Gy single doses of totalbody gamma-radiation. The experiment was performed as described aboveusing inactive flagellin fragment (CB) as a negative control.

As expected, we observed a 100% mortality in both groups at 17 and 20 Gy(FIG. 18). However, death was significantly delayed in both cases byadministration of the AA′ fragment. Most remarkably, the kinetics ofdeath at 17 Gy in control mice conform to GI syndrome (6-7 daymortality), while death of mice treated with the AA′ fragment appear tobe mediated by hematopoietic syndrome (10-15 day mortality). This showsthat flagellin and flagellin fragments may protect against the GIsyndrome at doses as high as 17 Gy. In addition, this shows that evenfurther radioprotection may be obtained by flagellin and flagellinfragments combined with hematopoietic radioprotectors.

Example 12 Immunogenicity and Repetitive Administration Studies

Overall immunogenicity of a protein may determines its suitability forrepeated use. Antibodies generated by immune system are capable ofreducing the therapeutic activity of the protein and also may induceanaphylactic reaction upon second exposure if IgE antibodies areproduced against the protein. Thus, any reduction in the amount andvariety of antibodies compared to full-length flagellin is animprovement. Accordingly, after repeated introduction of flagellin orits fragments we monitored; a) efficiency of radioprotection afforded atsecond exposure; b) local and general allergic reactions; and c)antibody titer.

We tested the ability of AA′ to protect mice that were exposed to it. Agroup of 20 NIH-Swiss mice were subcutaneously injected with 5 μg/mouse(0.2 mg/kg) of AA′. A second injection of a equal dose of AA′ wasadministered after 21 (10 mice) and 28 days (another 10 mice), with thetime elapsed being sufficient for formation of antibodies. The secondinjection of AA′ was followed by 13 Gy of whole-body gamma irradiation(1 h post-injection). 100% 30-day survival was observed in both groups,as it was observed with mice that had no previous exposure to AA′ (datanot shown). These results show that activity of AA′ is not diminishedover long-term repeated administration and reaffirm its potential formultiple-use applications. Also, no local allergic reaction oranaphylaxis was observed either with flagellin or AA′.

We also performed an ELISA determination of antibody titers in order toquantify the effect that AA′ has on the immune status of the organism.96-well plates were coated with flagellin or AA′, 20 mg/ml, 50 ml/well,and incubated overnight at +4° C. Blood serum samples collected frommice were added to the wells in several dilutions and incubatedovernight followed by 6 hrs reaction with secondary goat anti-mouse IgGHPO-conjugate antibodies. Measurements were performed using aspectrophotometer with a 414 nm filter. The antibody titers determinedfor individual mice and average titers are shown in FIG. 19 and FIG. 20.

AA′ induces far lower antibody levels in mice (FIG. 19), on the order of0.8 mg/ml serum at 21 day and about 10% more at 28 days. Flagellin, onanother hand (FIG. 20), induces a high titer of antibodies, around 20mg/ml, at both 21 and 28 days. Overall, this shows that removal of thehypervariable domain sharply reduces the immunogenicity of AA′ comparedto the original protein (approximately 25×). FIG. 19 also shows that themajority of AA′-specific antibodies are capable of recognizingflagellin. This confirms that the rational design of AA′ does notproduce a sizable number of new immunogenic epitopes while removing >95%of the immunogenicity of the original protein.

Example 13 Acute Toxicity Studies of Flagellin and AA′

The lethal dose of Salmonella flagellin is between 1 mg/kg (systemicinflammation) and 10 mg/kg (100% lethality). We subcutaneouslyadministered increasing doses of AA′ to mice (4 mice per dose group) at0.5, 1, 2, 4 and 8 mg/kg. Due to the lower (−60%) molecular weight, 8mg/kg of AA′ correspond to a molar-equivalent dose of 13.3 mg/kg offlagellin. Several days after administration at all doses, no visibledetrimental effects were observed, such as mortality, morbidity or signsof systemic inflammation such as reduced activity and fever. This showsthat the pro-inflammatory effect of AA′ is negligible compared tofull-length flagellin, especially considering that AA′ provides anefficient radioprotection at 0.2 mg/kg. The reduced toxicity may be sueto the absence of the central pro-inflammatory domain in the AA′fragment.

Example 14 Protection from Fractioned Irradiation by AA′

Repetitive irradiation within a short period of time may be common, forexample, in space radiation events and in clinical radiotherapyregimens. We tested the ability of the AA′ flagellin fragment to protectmice from sub-lethal (4 treatments of 3 Gy) and 100% lethal (4treatments of 4 Gy) regimens of fractionated gamma-irradiation. FragmentAA′ or saline buffer was given to NIH-Swiss female mice before everyirradiation (once a day for 4 days). AA′ was administered as describedabove for single-dose irradiation (5 μg/mouse, given subcutaneously 1 hbefore irradiation).

The results in FIG. 21 show that AA′ provides significant protectionagainst repetitive doses of radiation received within a short timeframe.The cumulative dose of fractionated radiation that is still compatiblewith 100% 30-day survival after AA′ treatment is comparable to suchobtained in single-dose irradiation scenarios.

Example 15 AA′ Protects Normal Tissues without Compromising theAnti-Tumor Therapeutic Effect of Radiation

The ultimate test of a potential radioprotective agent in cancertreatment is tumor selectivity, its ability to protect normal tissueswhile providing no or little protection to the tumor. We injected 10NIH-Swiss mice subcutaneously, in both flanks (20 tumors total), with2×10⁶ cells of syngeneic sarcoma cell line model (NIH3T3-derived andspontaneously transformed sarcoma with p53 inactivated by dominantnegative inhibitor GSE56). When tumors reached the size of 5-7 mm indiameter (day 5), the mice were injected subcutaneously with 0.2 mg/kgof AA′ or saline vehicle and irradiated 1 hours later with 4 Gy oftotal-body γ-irradiation (3×4.3 Gy=12.9 Gy total dose). Injections andirradiations were done at days 5, 6 and 7.

As the results show in FIG. 11, AA′ enhanced the radiation-inducedshrinkage of tumors. By day 18, all the irradiated tumor-bearing micedied from acute radiation toxicity whereas 100% of mice that obtainedboth radiotherapy and AA′ were both cured and survived the treatment.Similar result was obtained with another syngeneic tumor model—B16melanoma cells (FIG. 11, right panel). Surprisingly, even inunirradiated mice, AA′ administration caused a decrease in the growthrate of tumors. This may be due to AA′-induced immunostimulating, whichis known to be caused by other ligands of Toll-like receptors. Theseresults indicate that AA′ increases the tolerance of mice to radiationwith no effect on the radiosensitivity of two types of tumors, thusopening the possibility of combining radiotherapy with AA′ to improvetreatment outcome.

Example 16 Radioprotective Mechanisms of AA′ and LPS are Different

Lipopolysaccharide of gram-negative bacteria (LPS) is a ligand ofanother Toll-like receptor, TLR4. LPS is a strong inducer of NF-κB and asubsequent cascade of cytokines. LPS is known as a radioprotectivecompound, but its high toxicity makes its use unfeasible(radioprotective dose is very close to the lethal dose). One of themajor mechanisms underlying the radioprotection by LPS is the activationof cyclooxygenase 2 (COX-2) that, in turn, drives the synthesis ofGI-protective prostaglandins. The possibility that radioprotection byTLR5 also relies on COX-2 activity was tested by administering s.c. LPS(2 mg/kg), AA′ (0.2 mg/kg') or vehicle 1 hr before irradiation ofNIH-Swiss mice in combination with i.p injection of 1 mg/kg of NS398, asynthetic COX-2 inhibitor, or the corresponding vehicle. The mice werethen treated with 13 Gy of total-body γ-irradiation. NS398 completelyabolished LPS-mediated radioprotection but not the radioprotection ofAA′ (FIG. 12). This result shows that AA′ does not significantly rely onCOX-2 for its activity and induces radioprotection by a mechanismdifferent from the mechanism of LPS-mediated protection.

Example 17 AA′ Protects Multiple Mouse Strains

We have extensively confirmed that AA′ protects NIH-Swiss and ICR micefrom radiation. To confirm that the radioprotection activity of AA′ isnot confined to a few mouse strains, several additional strains of micewith dissimilar origins were tested for protection by AA′: 129/Sv, DBA/2(relatively radioresistant), Balb/c (relatively radiosensitive) andBalb/c×DBA/2 F1 hybrid CD2F1. Experimental groups were injected with 0.2mg/kg AA′ 30 minutes before irradiation, while control groups wereinjected with vehicle (PBS).

All groups of mice (8-10 mice each, 8-12 week old females) were exposedto 10 Gy of single-dose, whole-body gamma-irradiation. Survival of miceat days 10 and 30 is shown. The results are shown in FIG. 27 as a conegraph. At 10 days, only Balb/c mice display mortality, which isdrastically reduced by AA′ administration (0% vs. 100% survival). At day30, all tested strains display improved survival (0-25% vs. 50-100%)after AA′ administration.

Example 18 Pharmacokinetics of AA′

Pharmacokinetic parameters (effective concentration and the duration ofdrug the presence in the organism) may be important for route, dose andtime of drug administration. The pharmacokinetics of CBLB502 (AA′) werethus tested for four common routes of injection: intravenous (i.v.),subcutaneous (s.c.), intraperitoneal (i.p.) or intramuscular (i.m.). Aradioprotective dose of CBLB502 (AA′), 0.2 mg/kg, was injected in 12-15week old ICR mice and plasma samples were collected at the specifiedtimes after injection (at least 3 mice/point). The levels of CBLB502 inplasma were measured by sandwich ELISA using known concentrations ofCBLB502 spiked in the control ICR plasma for calibration. The resultsare shown in FIG. 28 and FIG. 29.

The results show that the highest levels and longest persistence ofCBLB502 in plasma are provided by intramuscular or intraperitonealinjection. After intramuscular injection, significant (>5 ng/ml) levelsof CBLB502 are observed in mouse plasma >3 hours. Intravenous injectionleads to a more rapid disappearance of CBLB502 from the bloodstream.

Example 19 Influence of AA′ on Gamma-Irradiation Induced Cell Death andGrowth Inhibition in A549 Cells

The A549 human lung adenocarcinoma cell line is reported to respond toflagellin by activation of NF-κB DNA-binding activity (Tallant T., et.al., BMC Microbiol. 2004 Aug. 23; 4:33). We decided to check whetherthis activation translates in the protection of cells from γ-IR in cellgrowth inhibition assay.

Tumor cells were seeded in wells of three 96-well plates in 3 differentdensities (0.5×10⁴, 1×10⁴ and 2×10⁴ cells/well, producing single-cell,spare or semi-contact layer). After cells had attached to plastic,CBLB502 (2 μg/ml) was added to the wells of non-irradiated cells, or 15min prior to 7 Gy or 10 Gy of gamma-irradiation. Control wells receivedequal volume of vehicle (PBS). All points were done in quadruplicate. 72hours after irradiation, medium was replaced with methylene blue in50%-methanol and the relative numbers of viable cells in wells weremeasured using spectrophotometer at 650 nm. The results are shown inFIG. 30. This experiment was also repeated with fixed dose of 1×10⁴ A549cells/well, CBLB502 was added 1 hour before 5, 10 or 15 Gy ofgamma-irradiation (data not shown). We observed a similar effect offlagellin in all tested experiment conditions.

Gamma-irradiation induced a dose-dependent reduction in the number ofA549 cells plated at all three densities (up to 60% as compared tonon-irradiated control wells). CBLB502 had no or slight effect on cellnumbers, with or without gamma-irradiation. This indicates that tumorcells are not significantly protected by CBLB502 (AA′) from radiation.This effect may be due to tumor cells having constitutively active NF-κBpathway or some other mechanism.

Example 20 Influence of AA′ on Gamma-Irradiation Induced Cell Death andGrowth Inhibition in Multiple Cell Lines

Based on the results using A549 cells, several additional tumor celllines (human melanoma Mel-7 and Mel-29, colon cancer HCT116, lung cancerHT1080), immortalized kidney epithelial cells (NKE) and normal mouseaortal endothelial cells (MAEC)) were tested in growth inhibition assayafter 10 and 15 Gy of gamma-irradiation, as compared with intactcontrol, with or without pretreatment with CBLB502. Cells were seeded in96-well plates night before the treatments. CBLB502 (2 μg/ml) was addedto the wells 4 hrs, 1 hr or 10 min before irradiation (all points weredone in quadruplicate). 48 hours later, methylene blue staining wasperformed to determine the relative amount of the viable cells in thewells. All three time-points had shown the same effect (results forCBLB502 added 1 hr before irradiation are shown in FIG. 31). The percentof growth inhibition was calculated from the OD650 in controlnon-irradiated wells, taken as 0% inhibition.

Both human melanoma cell lines and MAEC cells were rather resistant togamma-irradiation and showed only slight (<20%) growth inhibition afterboth 10 and 15 Gy comparing with intact cells (0 Gy). NKE, HT1080 andHCT116 cells showed up to 40% of growth inhibition aftergamma-irradiation. Remarkably, CBLB502 had no or only a slightinhibitory effect on tumor cell growth, irradiated or not. Theexperiment was repeated twice. In addition, similar results wereobtained on tested lung adenocarcinoma H1299 and prostate cancer CWR22(data not shown). This indicates that there is no significant protectionprovided by CBLB502 to the tumor cell lines against radiation-inducedcell death.

Example 21 Influence of Irradiation and AA′ on BrdU Incorporation inSmall Intestinal Crypts

Besides a direct inhibition of apoptosis, temporary halt ofproliferation followed by repair may be an alternative mechanism ofradioprotection, and has been described for other radioprotectors suchas TGF-β3 (Booth D., et. al., Int J Cancer. 2000 Apr. 1; 86(1):53-9).Accordingly, we decided to examine the possible influence of CBLB502(AA′) on the proliferative activity of the cells in small intestine(with and without irradiation) during the first hours after itsadministration (FIG. 32). CBLB502 or PBS was injected i.p. in mice,followed after 30 min by 15 Gy irradiation (if used). 2 hr afterinjection (1.5 hr after irradiation if it was applied), BrdU wasinjected intraperitoneally. Samples of small intestine were obtainedafter additional 1.5 hours.

Without irradiation, BrdU was incorporated at high levels in the nucleiof cells in the intestinal crypts of untreated NIH-Swiss mice (FIG. 32,top left), whereas DNA synthesis (as measured by BrdU incorporation) wasnearly undetectable in the crypts of CBLB502-treated mice (FIG. 32, topright). In vehicle-treated irradiated mice, the incorporation of BrdUwas lower than in control mice. Importantly, the level of BrdUincorporation was strongly reduced by CBLB502, possibly indicating quick(S phase) growth arrest, as opposed to later (G2 phase)irradiation-induced growth arrest. Therefore, cytostatic activity ofCBLB502 or flagellin may be an additional mechanism of radioprotectionof small intestine.

Example 22 Duration of AA′-Mediated Growth Arrest and Reduced BrdUIncorporation

We next determined the duration of the CBLB502-induced growth arrest insmall intestine. CBLB502 or PBS was injected i.p. in mice, BrdU wasinjected 1 or 4 hours later and samples of small intestine were obtainedafter additional 1.5 hours from several mice (samples from three miceare shown) (FIG. 33).

Incorporation of BrdU in intestine was reduced as compared to control ifBrdU was injected after 1 hour (as it was shown in the previousexperiment where BrdU was injected after 2 hr). NIH-Swiss, ICR andBalb/c mice displayed a similar degree of CBLB502-mediated block of BrdUincorporation (Balb/c samples are shown in FIG. 33). If BrdU wasinjected 4 hr after injection of CBLB502, the levels ofincorporation/DNA synthesis were even higher than in control. Thisindicates that inhibition of intestinal stem cell proliferation byCBLB502 may be temporary and may be quickly resolved (by 4 hours),followed by a period of increased proliferation (possibly due to thepartial synchronization of cells).

Example 23 Influence of AA′ on BrdU Incorporation in Colonic Crypts

The colon is much less radiosensitive than small intestine. To furtherexamine the relationship between reduced proliferation in the smallintestine and radioprotection, we determined the effect of CBLB502 onBrdU incorporation in the colon. CBLB502 or PBS was injected i.p. inmice, BrdU was injected 1 hour later and samples of small intestine wereobtained after additional 1.5 hours (FIG. 34).

Unlike in the small intestine, CBLB502 has no effect on BrdUincorporation in colon. This is surprising since TLR5 is plentiful inboth organs. The difference in effects may be due to the higher amountof symbiotic bacteria in the colon, which may mask the effect ofadditional TLR5 signaling induced by CBLB502.

Example 24 Comparison of Radioprotective Potential by Route ofAdministration

We next tested radioprotection provided by FliC flagellin administeredvia several routes: intravenous (i.v.), intraperitoneal (i.p.),intramuscular (i.m.), subcutaneous (s.c.) and gavage. For parenteral(non-gavage) routes, mice were injected with 0.2 mg/kg of FliC flagellindissolved in PBS or vehicle, followed 1 hr later by 13 Gy irradiation.In gavage delivery experiment, 5 mice were given to swallow an increaseddose (50 μg) of FliC in 50 μl of PBS 1 h before 13 Gy gamma-irradiation.Both experiments were done in 8-10 week old female NIH-Swiss mice, 5-10mice/group.

All tested routes besides gavage afforded similar degree of protection,leading to 85-90% 30-day survival of mice (data not shown). Noprotection against radiation was provided by gavage delivery, which maybe due to digestion of the protein by the gastrointestinal environment.In addition, flagellin receptor, TLR5, is absent on the luminal side ofintestinal epithelium that is exposed to intestinal contents (Gewirtz AT., et. al., J Immunol. 2001 Aug. 15; 167(4):1882-5)

Example 25 Effect of AA′ on the Morphology of Small Intestine

Flagellin (and CBLB502) may induce NF-κB activity via binding to TLR5.Accordingly, CBLB502-mediated radioprotection may be dependent on thepresence and activity of TLR5. MOLF/Ei mice are a known natural model ofTLR5 deficiency (Sebastiani G., et. al., Genomics. 2000 Mar. 15;64(3):230-40). To verify that CBLB502-mediated radioprotection is indeedTLR5-dependent, we tested the protection of small intestine fromradiation by CBLB502 in MOLF/Ei and NIH-Swiss mice (FIG. 35). Bothstrains of mice were given 0.2 mg/kg CBLB502 (AA′) or PBS 0.5 hr before15 Gy of gamma-irradiation. The samples of small intestine were obtained4 days after irradiation, stained by hematoxylin-eosin and subjected topathomorphological analysis.

In NIH-Swiss (TLR5 wild type) mice, CBLB502 pretreatment led topreservation of intestinal morphology (long villae, normal crypts) ascompared to short villae and disappearance of normal crypt structure inPBS-treated mice. Meanwhile, in TLR5-deficient MOLF/Ei mice theadministration of CBLB502 had no improving effect on intestinalmorphology after 15 Gy of gamma-irradiation: short villae anddestruction of the normal crypt structure was observed, with or withoutCBLB502. This indicates that the presence of TLR5 may be necessary forCBLB502-mediated radioprotection in the small intestine.

Example 26 Flagellin Derivatives

Additional flagellin variants were produced based on the domainstructure shown in FIG. 36. The flagellin variants were then testedalong with some of the variants discussed above for NF-κB stimulatingactivity (Table 3). A549 cells were left unstimulated or stimulated withTNF (10 ng/ml) as indicated or 1 μg/ml of purified flagellin or thevarious indicated flagellin derivatives for 45 min and whole cellextracts prepared as described in Example 7. EMSA assays were preformedand the NF-κB DNA-protein complex detected as described in Example 7.

TABLE 3 NF-κB Name N-terminal C-terminal DNA Protein Stimulation AA′ 1-176 402-505 SEQ ID NO: 7 SEQ ID NO: 8 Yes AB′  1-176 402-450 SEQ IDNO: 9 SEQ ID NO: 10 Yes BA′ 54-176 402-505 SEQ ID NO: 11 SEQ ID NO: 12Yes BB′ 54-176 402-450 SEQ ID NO: 13 SEQ ID NO: 14 No CA′ 54-100 402-505SEQ ID NO: 15 SEQ ID NO: 16 No CB′ 54-100 402-450 SEQ ID NO: 17 SEQ IDNO: 18 No AA′n1-170  1-170 402-505 SEQ ID NO: 29 SEQ ID NO: 30 YesAA′n54-170 54-170 402-505 SEQ ID NO: 31 SEQ ID NO: 32 Yes AB′n1-170 1-170 402-450 SEQ ID NO: 37 SEQ ID NO: 38 Yes AA′n1-163  1-163 402-505SEQ ID NO: 33 SEQ ID NO: 34 Yes AA′n54-163 54-163 402-505 SEQ ID NO: 35SEQ ID NO: 36 Yes AB′n1-163  1-163 402-450 SEQ ID NO: 39 SEQ ID NO: 40Yes AA′n1-129  1-129 402-505 SEQ ID NO: 41 SEQ ID NO: 42 Yes AA′n54-12954-129 402-505 SEQ ID NO: 43 SEQ ID NO: 44 Yes AB′n1-129  1-129 402-450SEQ ID NO: 45 SEQ ID NO: 46 untested AB′n54-129 54-129 402-450 SEQ IDNO: 47 SEQ ID NO: 48 Untested AA′n1-100  1-100 402-505 SEQ ID NO: 49 SEQID NO: 50 untested AB′n1-100  1-100 402-450 SEQ ID NO: 51 SEQ ID NO: 52untested AA′n1-70 1-70 402-505 SEQ ID NO: 53 SEQ ID NO: 54 No AB′n1-701-70 402-450 SEQ ID NO: 55 SEQ ID NO: 56 No A  1-176 SEQ ID NO: 19 SEQID NO: 20 No B 54-176 SEQ ID NO: 21 SEQ ID NO: 22 No C 54-100 SEQ ID NO:23 SEQ ID NO: 24 No GST-A′ 402-505 SEQ ID NO: 25 SEQ ID NO: 26 No GST-B′402-450 SEQ ID NO: 27 SEQ ID NO: 28 No

The results in Table 3 indicate that flagellin variants with at leastone polymerization domain (aa1-50 or aa 450-505) that linked to domainscontained within the amino-terminal region (aa 1-176) and those of thecarboxy terminus (aa 402-505) are capable of stimulating NF-κB and wouldthus be expected to be radioprotectors. Physical linkage of therecognition domains may be required for activity as domains suppliedunlinked in trans fail to activate NF-κB. As an alternative to thelinking of the domains in a single polypeptide, the domains may belinked using a linker, which is a molecule that is used to join twomolecules. The linker may be capable of forming covalent bonds orhigh-affinity non-covalent bonds to both molecules. Suitable linkers arewell known to those of ordinary skill in the art and include, but arenot limited to, straight or branched-chain carbon linkers, heterocycliccarbon linkers, or peptide linkers. The linkers may be joined to theconstituent amino acids through their side groups (e.g., through adisulfide linkage to cysteine).

The region between amino acids 163 and 176 may be required for activitywhen the carboxyl polymerization domain (aa 450-505) is absent. Sincethis region is dispensable for activity when the carboxyl polymerizationdomain is present it may be involved in stabilizing the derivative. Theregion between amino acids 70 and 129 may be important for activationand may be involved in derivative recognition. The region between aminoacids 402 and 450 may also be required for activity. The domainsidentified above are located within three large α-helices (locatedwithin amino acids 54-129 and 402-450) and, to produce an activederivative, may need to form a ring-like structure (with or withoutpolymerization domain).

1.-13. (canceled)
 14. A composition comprising a flagellin polypeptide,wherein the polypeptide comprises a non-antigenic linker operativelylinked to the D₀ amino terminal domain and the D₀ carboxyl terminaldomain.
 15. The composition of claim 14, wherein the sequence of the D₀amino terminal domain comprises: (a) amino acids 1-54 of SEQ ID NO: 1;or (b) a sequence at least 70% identical to (a).
 16. The composition ofclaim 15, wherein the sequence of the D₀ carboxyl terminal domaincomprises: (a) amino acids 451-505 of SEQ ID NO: 1; or (b) a sequence atleast 70% identical to (a).
 17. The composition of claim 14, wherein theflagellin polypeptide comprises a sequence selected from the groupconsisting of SEQ ID NOS: 10, 12, 30, 32, 34, 36, 38, 40, 42, 44, 46,48, 50, and 52.